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Rho Kinase (ROCK) Inhibitors and Their Therapeutic Potential Yangbo Feng, Philip V. LoGrasso, Olivier Defert, and Rongshi Li J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b00683 • Publication Date (Web): 20 Oct 2015 Downloaded from http://pubs.acs.org on October 23, 2015

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Journal of Medicinal Chemistry

Rho Kinase (ROCK) Inhibitors and Their Therapeutic Potential

Yangbo Feng,‡,* Philip V. LoGrasso, ‡,¥ Olivier Defert,± and Rongshi Li†,*

‡

Translational Research Institute, ¥Department of Molecular Therapeutics, The Scripps Research

Institute, Florida, 130 Scripps Way, #2A2, Jupiter, FL 33458. ±

†

Amakem Therapeutics, Agoralaan A bis, 3590 Diepenbeek, Belgium

Center for Drug Discovery and Department of Pharmaceutical Sciences, College of Pharmacy,

Cancer Genes and Molecular Regulation Program, Fred and Pamela Buffett Cancer Center, University of Nebraska Medical Center, 986805 Nebraska Medical Center, Omaha, NE 68198.

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Abstract Rho kinases (ROCKs) belong to the serine-threonine family, the inhibition of which affects the function of many downstream substrates. As such, ROCK inhibitors have potential therapeutic applicability in a wide variety of pathological conditions including asthma, cancer, erectile dysfunction, glaucoma, insulin resistance, kidney failure, neuronal degeneration and osteoporosis. To date, two ROCK inhibitors have been approved for clinical use in Japan (Fasudil and Ripasudil) and one in China (Fasudil). In 1995 Fasudil was approved for the treatment of cerebral vasospasm and, more recently, Ripasudil was approved for the treatment of glaucoma in 2014. In this Perspective, we present a comprehensive review of the physiological and biological functions for ROCK, the properties and development of over 170 ROCK inhibitors as well as their therapeutic potential, the current status and future considerations.

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1. Introduction 1.1. Current kinase inhibitor drugs on the market. Following the discovery of protein kinases in 19541, it was almost 50 years before the launch of Gleevec (1, imatinib)2, the first selective tyrosine kinase inhibitor approved for the treatment of multiple cancers including chronic myelogenous leukemia (CML). Since the publication of a comprehensive review of the discovery of the first eight (and several more advanced) kinase inhibitor drugs in 20093, 19 additional smallmolecule protein kinase inhibitor drugs (structures shown in Figure 1) have been approved by U. S. Food and Drug Administration (FDA). Despite the various targets of these molecules, all kinase inhibitor drugs share the same enzymatic mechanism i.e. inhibition of the transfer of the terminal phosphate from ATP to their substrates. Within the human genome 518 protein kinases are encoded4, and the “kinome” has been catalogued according to sequence similarity in the catalytic domain. Inhibitors of the protein kinase family (including tyrosine, serine and threonine, and histidine kinases) are classified into four types based on their mechanism of action. Most kinase inhibitors are type-I, i.e. those targeting the ATP binding site of the kinase in its active conformation, reversibly competing with ATP. There are nine known type-II kinase inhibitors (1, 4, 5, 8, 9, 16–19) that stabilize an inactive form of the kinase and adopt a DFG-out conformation in an activation-loop with high selectivity5-7. Although the study of highly selective allosteric kinase inhibitors (type-III) is an active area of research8, 9, only one drug (22) has been approved to date10. Type-IV kinase inhibitors are covalent kinase inhibitor drugs (20 and 21). 1.2. ROCK (Rho kinase) signaling pathways with downstream targets and direct biological regulations. Rho is a small GTPase that can be activated by guanine nucleotide exchange factors (GEF). GTP-bound RhoA subsequently activates ROCKs to phosphorylate a variety of sub-

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strates9 as shown in Figure 2. For example, phosphorylation of the myosin binding subunit of myosin light chain phosphatase (MLCP) by ROCK inhibits the dephosphorylation of myosin light chain (MLC), which mediates actin cytoskeletal changes. ROCK also can directly phosphorylate MLC. The downstream LIMK kinases (LIM kinases 1 and 2) are involved in regulation of actin-filament dynamics. Myristylated alanine-rich C-kinase (MARCKS), neurofilament protein (NF-L), collapsing response mediator protein-2 (CRMP2), adducin, sodium/hydrogen exchanger 1 (NHE1) and the ezrin-radixin-moesin (ERM) family of actin-binding proteins are all downstream substrates that lead to cellular responses and cytoskeletal regulation. ROCK is a member of the AGC (cAMP-dependent protein kinase/protein kinase G/protein kinase C) kinase family. Two isoforms, ROCK-I and ROCK-II, were isolated as RhoA-GTP interacting proteins of ~160 kDa11. The amino acid sequence of the two ROCK isoforms share ~60% of their overall identity and ~90% in their kinase domain12, 13. ROCK inhibitors have high potentials for the treatment of a variety of pathological conditions including asthma14, cancer15, erectile dysfunction16, glaucoma17, insulin resistance18, kidney failure19, neuronal degeneration20 and osteroporosis17. To date, only two ROCK inhibitors are approved for clinical use, in Japan and/or in China: Fasudil (28, Figure 3) was approved in 1995 for the treatment of cerebral vasospasm, and Ripasudil (165, Figure 16) was approved in 2014 for the treatment of glaucoma. In this Perspective, the physiological and biological functions of ROCK inhibitors, their development, therapeutic potential, and the current status are reviewed. 2. Physiological and Biological function of Rho Kinases 2.1. Rho kinase isoforms and ROCK-I/ROCK-II isoform selectivity. Two isoforms of Rho kinase have been described. Human ROCK-I (also referred to as p160 ROCK or ROKβ)21 and hu-

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man ROCK-II (ROKα)13 are approximately 160-kDa proteins containing an N-terminal Ser/Thr kinase domain, followed by a coiled-coil structure, a pleckstrin homology domain and a cysteine-rich region at the C-terminus. The two isoforms share approximately 60% overall amino acid identity and approximately 90% identity within the N-terminal kinase domain12, 13. Importantly, there is 100% identity within the ATP binding pocket between the two isoforms accounting for the paucity of isoform selective inhibitors. In 1996 Narumiya and colleagues12, 21 reported the discovery of ROCK-I and ROCK-II and showed by Northern blot analysis that ROCK-I was expressed in many human tissues including the heart, pancreas, lung, liver, skeletal muscle, and kidney, but not the brain21. In their investigation, they showed ubiquitous mRNA expression of both ROCK-I and ROCK-II in all mouse tissues, including the brain, with ROCK-I preferentially expressed in the liver, spleen and kidney, and ROCK-II preferentially expressed in the brain and skeletal muscle12. In 1999, Takahashi et al. reported the isolation of the human ROCK-II isoform from a brain cDNA library13. Thus, it is generally regarded that both ROCK isoforms are ubiquitously expressed with perhaps a greater expression of ROCK-II in the brain compared to ROCKI. In addition to sharing high sequence identity, the two isoforms also share many protein substrates including myosin light chain (MLC)22-24, myosin light chain phosphatase (MYPT1)25, 26, LIM Kinases (LIMK)27-29, and S6 peptide30-33 suggesting some redundancy in their biological roles. Given the sequence identity of the two ROCK isoforms in the ATP pocket, the substrates that both isoforms share, and the ubiquitous expression of each isoform, it is necessary to look for possible differentiators in function for these two isoforms. In an effort to study ROCK-II function, Narumiya and colleagues generated ROCK-II knockout mice34. This targeted disruption produced striking results: 90% of the embryos died in utero and the surviving pups were born as

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runts. For the most part, the survivors developed normally and were fertile and embryonic lethality was attributed to defects in the embryo and placenta interaction where there was marked thrombosis and loss of trophoblasts in the labyrinth layer. Similarly, disruption of the ROCK-I gene showed developmental problems manifesting in two prevalent phenotypes: eyes open at birth (EOB) and ophalocele in neonates35. This was attributed to disorganization of the actomyosin cables in the eyelid and defective actin assembly in the umbilical ring. Importantly, these different phenotypes demonstrated that ROCK-I and II could not compensate for each other during early development. In a follow-up study the same investigators, utilizing a backcross of ROCK-II mice (mixed 129/Sv and C57BL/6N) into a C57BL/6N background, found that both ROCK-I and ROCK-II cooperatively regulate the assembly of actin bundles affecting eyelid closure and ventral body wall in the developing mouse embryo36. Interestingly, Wei and colleagues published a series of papers37-39 describing ROCK-I deletions utilizing a different genetic background than the Narumiya group. In that study, the EOB and omphalocele phenotypes were not manifested, suggesting different effects in different genetic backgrounds. These results highlight some of the limitations in using genetic models to study the role of individual isoforms and emphasize the importance and value of a pharmacological approach where highly selective isoform inhibitors are used to define each isoform’s role. In addition to the knockout mouse models described above, numerous attempts have been made to utilize ROCK-I (-/-) and ROCK-II (-/-) mouse embryonic fibroblasts (MEFs) to understand the individual role each isoform may play in different cell types. Noguchi et al. demonstrated that adipogenesis of ROCK-II (-/-) (MEFs) was enhanced compared to wild type MEFs, whereas no change was seen in ROCK-I (-/-) MEFs. Moreover, ROCK-II RNAi experiments showed enhanced expression of adipogenic transcription factors and ROCK-I knockdown did not

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suggest a unique role for ROCK-II in adipogenesis40. In a recent 2013 study, Shi et al. utilized ROCK-I (-/-) and ROCK-II (-/-) MEFs to dissect the role of individual ROCK isoforms in regulating actin cytoskeleton reorganization in response to doxorubicin treatment. The ROCK-I (-/-) MEFs showed improved cytoskeleton stability and decreased MLC2 phosphorylation, but had no effect on cofilin phosphorylation. These changes resulted in cell shrinkage, detachment, and apoptosis. In contrast ROCK-II (-/-) MEFs showed impaired cell adhesion with decreases in both MLC2 and cofilin phosphorylation. The authors concluded that ROCK-I was responsible for destabilizing actin cytoskeleton and regulating MLC phosphorylation, whereas ROCK-II was responsible for stabilizing actin cytoskeleton through regulating cofilin phosphorylation41 suggesting unique contrasting roles for the two isoforms. In a follow-up study the same group showed that both ROCK-I and ROCK-II (-/-) MEFs reduced reactive oxygen species (ROS) production in response to doxorubicin, but only ROCK-I (-/-) MEFs worked in concert with Nacetylcysteine in reducing ROS42. While these studies, like the knockout mice, shed some insight on the individual roles of the two ROCK isoforms, they only begin to scratch the surface in our understanding of the potential physiological and biochemical differences between the two isoforms. 2.2. Structural Biology of ROCK. At the biochemical and structural level, there has been little differentiation between these two isoforms. Ten crystal structures of ROCK-I have been deposited in the protein data bank (www.rcsb.org/pdb) and three for ROCK-II. Of those ten ROCK-I structures, five were complex structures with 28 (Figure 3) or its derivatives hydroxyfasudil (29), dimethylfasudil (30) and other isoquinoline based analogs. Most of these structures were solved at moderate resolution ranging from 2.96Å – 3.30Å. Jacobs et al. reported the crystal structure of human ROCK-I residues 6-415 and showed that this protein crystallized as an N-terminal head-

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to-head dimer43. Jacobs et al. presented four other structures, three of which were of analogs of 28, and one from the pyridine carboxamide, Y27632 (31, Figure 3). One of the key conclusions from that work described A215 in ROCK-I (T183 in PKA) as the main determinant for selectivity of these compounds for ROCK-I over PKA43. In 2006, Yamaguchi et al. published two papers reporting the 2.4 Å and 3.1 Å crystal structures of bovine ROCK-II residues 18-41744, 45. Here too the authors found an N-terminal head-to-head dimeric structure. The 2.4 Å co-crystal structure of 28 in bovine ROCK-II contains two crystallographically independent molecules (A and B)44. In both molecules 28 binds to the ATP pocket with the isoquinoline nitrogen atom H-bonding to hinge residue M172 and the sulfonamide moiety pointing toward the catalytic loop44. The free NH group of the 7-membered diazepane ring forms H-bonds in both molecules but with different surrounding residues44. In molecule A, the pendant diazepane ring is twisted to an area under the P-loop and the NH group is sitting around the DFG motif and forms two H-bonds with residues N219 and D232. In molecule B, the diazepane ring is aligned perpendicular to the isoquinoline ring and is pointing toward the catalytic loop (C-loop). The free NH group of the diazepane ring in molecule B forms two H-bonds with residues D176 and D21844. Interestingly, the P-loop in molecule B flips down toward 28, and the aromatic ring of F103 (on the P-loop) makes contact with the diazepane ring of 28 (thus stronger hydrophobic interactions that can further facilitate ligand binding). In addition, the carbonyl group of F103 in molecules B is H-bonded to the side chain of the catalytic residue lysine K121 that also forms Hbonds or ion pairs with residues E140 and D232 of the DFG motif44. The 3.1 Å co-crystal structure of 31 in ROCK-II45 indicates that the pyridine nitrogen atom is H-bonded to hinge residue M172. The cyclohexane ring is pointing toward the P-loop with the primary amino group NH2 forming two H-bonds with residues N219 and D232 of the DFG mo-

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tif. Interestingly, this crystal structure also revealed an induced-fit binding mode and it was suggested that structural differences found in the conformation of the P-loop residue F103 appeared relevant to inhibitor binding. Specifically it was suggested that F103 turned inward in ROCK-II (similar to that in molecule B of the crystal structure of 28 in ROCK-II44), but (F87 in ROCK-I) projected outward in ROCK-I45. Given the modest resolution of all of the isoquinoline structures and the structural similarity of all those compounds, not a great deal of information can be garnered for discerning how one might achieve isoform selective ROCK inhibitors. Even with the higher resolution structure (2.60Å) for 31 (a non-isoform selective inhibitor), Jacobs et al. did not reveal any insights as to how one might achieve isoform selective ROCK inhibitors. Since the publication of the original crystal structures, numerous novel scaffold ROCK inhibitors have been reported (for a review of those classes up to 2009, see LoGrasso et al46, and for more recent patent applications of 2012-2013, see Feng et al47). Since then, many classes of compounds30-32, 48-52 and a number of new crystal structures have been revealed. One is the crystal structure of human ROCK-I complexed with pyridylthiazole urea-based inhibitors53. In that report, the structure of human ROCK-I and inhibitor RKI-1313 [1-(4-methoxybenzyl)-3-(4(pyridin-4-yl)thiazol-2-yl)urea]53 was solved at 2.75Å and showed typical type-I kinase inhibitor binding, having hydrogen bonding interactions with the hinge region, the hydrophobic core and hydrogen bond interactions between the urea of the inhibitor and D216 of the DFG segment. A more potent analog RKI-1447 (170, Figure 16)53 was also reported and the crystal structure showed a similar binding mode with additional hydrogen bonding to G85 of ROCK-I. This additional hydrogen bonding and the loss of steric hinderance from the original inhibitor were described as the likely reason for the increased potency. A higher resolution structure solved for ROCK-I at 2.30Å was reported in 2012 by Li et al54. In that report, a novel urea-indazole-based

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inhibitor 1-(1H-indazol-5-yl)-3-phenethylurea (42, Figure 4) was shown to have many similar binding features to other classes of compounds where hydrogen bonding interactions were found between the urea nitrogen atoms and D216 in the hinge region. The most recent crystal structure for ROCK-I was reported by Green et al.55 The pyridine based inhibitor 2-(3-methoxyphenyl)-N(4-(pyridin-4-yl)thiophen-2-yl)acetamide (58, Figure 5), complexed with ROCK-I, revealed that the pyridine moiety bound to the hinge region forming an H-bond with residue M156, the amide C=O was H-bonded to the side chain of K105, and a third H-bond was formed between the 3’methoxy group and the backbone NH of residue F8755. It is interesting to note that, in that crystal structure, the hinge binding pyridine, the central thiophene ring and the amide -CONH- moiety all assumed a flat conformation55. Moreover, the terminal benzyl group flipped upwards (~ 100º) and bound to the P-loop with the methoxy oxygen H-bonding to the backbone of the enzyme. Of the ROCK-II structures reported recently, one is for the human ROCK-II56. In that study ROCK-II was co-crystallized with a modestly potent benzoxaborole inhibitor (AN3484, 131, Figure 12) at a 2.79Å resolution. Like the indazole urea and 31, this benzoxaborole shared many binding features with other compounds. One unique feature for the benzoxaborole was a fluorine atom filling a pocket created by V106 and K121. The second crystal structure for ROCK-II was reported by Boland et al.57 In that structure, a highly potent (ROCK-II, IC50 = 2 nM) and softdrug based inhibitor (144 in Figure 13) was complexed to ROCK-II and the structure was resolved at a 2.93Å resolution57. Compound 144 is a derivative of Y32885 or Wf536 (33, Figure 3), an analog of 31. The H-bonding interactions observed in the 31-ROCK-I complex43 were also found in that crystal structure. The much higher potency for 144 (vs. 31) results mainly from the added hydrophobic interactions and three extra H-bonding interactions: one is between the ester and the side chain of T235; the other two are between the bi-phenyl benzamide and residues

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F103 and D23257. Moreover, the two additional phenyl groups in 144 provide added hydrophobic interactions with the P-loop and π-stacking interactions with F13657. Given the similarities between crystal structures of ROCK-I and ROCK-II, the question of which structural features give rise to isoform selective ROCK inhibitors, and how these are manifested in a functional physiological way, remains unanswered. For example, ROCK-II isoform selective inhibitors might find wide applications in the central nervous system (CNS), such as for treating stroke58, 59, Alzheimer’s disease (AD)60, 61, spinal cord injuries (SCI)62, 63, multiple sclerosis (MS)64, and neuroblastoma65, 66, etc. In addition, the hypotensive effects of systemic ROCK inhibition is speculated to be mainly due to ROCK-I inhibition47. Therefore, ROCK-II isoform selective inhibitors might provide drugs with less safety concerns. 3. Development of ROCK Inhibitors and Their Therapeutic Implications Over the past two decades, numerous ROCK inhibitors have been developed from a variety of distinct scaffolds. Many of these inhibitors20, 46, 47, 67-74 and/or their biological functions11, 20, 69, 72, 75-86

have been reviewed. Almost all ROCK inhibitors disclosed to date are ATP competitive

kinase inhibitors5, 87. In this Review, we will discuss ROCK inhibitors primarily based on their hinge binding moieties. Our focus will be on their potency, selectivity, binding modes, drug metabolism/pharmacokinetics (DMPK) and the biological functions. 3.1. Classic ROCK inhibitors. One type of classic ROCK inhibitors are the isoquinoline based 28 (Figure 3) and its derivatives. Compound 28 is not only one of the first kinase inhibitors, it is also the first kinase inhibitor that has advanced to clinical use in Japan and in China. 28 had a moderate ROCK potency with a Ki of ~ 0.33 µM against ROCK-II88. Crystal structures complexed with ROCK-I and ROCK-II showed that the isoquinoline nitrogen bound to the hinge region43, 44. The in vivo activity of 28 was reported to be a combined effect of both the parent

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molecule and its metabolite 2989, 90, which had a similar potency to 28 but a slightly different binding mode43. Further optimization of 28 led to the much more potent 30, which was reported to have a Ki value of 1.6 nM (Figure 3)91, 92. Both 28 and 30 were reported to have only moderate kinase selectivity93, and had inhibitory activity against a number of other kinases in the AGC family with 30 being more selective92, 94. Nevertheless, 28 and 30 are still widely used as a probe to study the biology related to Rho kinase pathways69, 95. Another classic ROCK inhibitor is 31 (Figure 3). Compound 31 and its derivatives are based on the 4-amidopyridine (and 4-amidopyrrolopyridine) scaffold and were first developed by Yoshitomi Pharmaceutical96, 97. 31 had a slightly higher potency than 28 with a reported Ki of 0.14 µM against ROCK-II98, 99, and its IC50 value against ROCK-II was reported to be in the range of 50 to 200 nM57, 98, 99. Interestingly, the pyrrolopyridine analog (32, Figure 3) of 31 was much more potent (Ki of 32 is 0.01 µM vs. 0.14 µM for 31), most likely due to a larger aromatic surface buried in the active site and the extra H-bonding interactions from the pyrrolopyridine NH moiety. However, this analog was less selective against PKA96, 98. The 4-benzamide counterpart of 31 (33, Figure 3) is also a moderate ROCK inhibitor with a ROCK-II IC50 of 200 nM100-103. The corresponding pyrrolopyridine derivative 34 is a remarkably potent inhibitor with an IC50 of only 3.6 nM for ROCK II103. Like 28, 31 was reported to show some selectivity against a number of kinases including PKC, PKA, etc.103, but its general kinase selectivity was revealed to be only moderate93. In addition, 31, 33, and Y39983 (34, Figure 3) were effective at reducing intraocular pressure (IOP) in rat and monkey eyes104-107 and had potential for treating other eye diseases108, 109

. An interesting class of secondary amine-based structures 35 and 36 (Figure 3) were disclosed

in a patent application110 by Devgen NV. These compounds were originally developed as PKC

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inhibitors, but it was speculated that they also had ROCK inhibition activity by comparing their structures to 33. 3.2. Indazole based ROCK inhibitors. Indazole derivatives can also be classified as classic ROCK inhibitors because they belong to one of the first few scaffolds developed as ROCK inhibitors in the late 1990s and early 2000s. Scientists from Kirin reported a series of 5-amino- or 5-alkoxy-indazole derivatives (37, Figure 4) as ROCK inhibitors111-113. Generally, the indazole moiety in these compounds function as the hinge binder, while the terminal aromatic group binds to the hydrophobic pocket under the P-loop. In the original Kirin structures, the terminal aromatic ring was normally a benzyl-type moiety. The “linker” in structure 37 could be an amide, a urea, a carbamate, or a simple carbocyclic or heterocyclic ring. The best compounds reported were shown to have a high ROCK potency with IC50 values in the single- to two-digit nM range. For example, the urea based compound 38 had an IC50 of 20 nM for ROCK-II, while the 3-ylpiperidine based inhibitor 39 was disclosed to have an IC50 value of 3 nM. Similar ROCK inhibitors based on this 5-substituted indazoles scaffold were also released in several patent applications from Inspire Pharmaceuticals114, 115. Feng et al. of the Scripps Research Institute extended this 5-aminoindazole scaffold to potent ROCK inhibitors composed of amino acid derivatives31. For example, the phenylglycine based amides 40 and 41 (Figure 4) exhibited IC50 values of 13 nM and 90 nM for ROCK-II, respectively. Li et al. at the Moffitt Cancer Center created novel compounds to contain the indazole-urea-phenethylamine moiety (42)54, 116. Structure-guided design and X-ray crystallography yielded ROCK inhibitor 42 and a few analogs using high concentration biochemical assays and fragment-based screening54. Among this class of ROCK inhibitors, compound 42 and a few analogs were shown to inhibit the phosphorylation of myosin light

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chain-2 (MLC-2) in the breast cancer cell line MDAMB-231.54 They also exhibited antimigratory and anti-invasive activities117. A series of 5-amido-indazole based ROCK inhibitors were reported by GlaxoSmithKline Pharmaceutical (GSK)118-123. As shown in Figure 4, compound 43 had an additional 6-membered ring molded between the 5-amido C=O and the terminal aromatic moiety, which resulted in a more rigid structure compared to Kirin’s urea based ROCK inhibitor 38. In addition, the extra amide group (or urea moiety for some compounds, structures not shown) in the central ring can offer additional H-bonding interactions. GSK’s indazole scaffold provided multiple compounds that exhibited higher potency with IC50 values in the single-digit nM range for ROCK-I118, 119. Furthermore, introduction of a fluorine substitution at 6-position of the indazole core (43) was necessary in order to improve in vivo pharmacokinetic properties. Thus, inhibitor 43 (Figure 4) had an IC50 of 14 nM for ROCK-I, a cell based IC50 of 190 nM in the rat aorta, and an oral bioavailability of 61% in rats118. Compound 43 was also demonstrated to reduce the mean arterial pressure in spontaneously hypertensive rats (SHR) in a single oral dose of 3 mg/kg118. Lee and colleagues from the Korea Research Institute of Chemical Technology (KRICT) reported DW1865 (44)124 as a potent ROCK-II inhibitor. Instead of a saturated ring as the linker in the Kirin compound 39, an unsaturated 1,3,5-triazine ring was connected to the 5-aminoindazole core in 44. Importantly, a terminal aromatic ring was not present in 44 but it still demonstrated a high ROCK-II potency, with an IC50 = 20 nM. This high potency is likely due to the H-bonding interactions between the terminal piperazine NH and the side chain carboxylate group of residue Asp176 in ROCK-II, as well as the hydrophobic interactions between the triazine ring and the hydrophobic pocket under the P-loop124. Inhibitor 44 was shown to be selective against 13 other

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kinases but no profiling data against a large kinase panel were provided124. In addition, 44 was demonstrated to have good cell potency in actin stress fiber formation assays, and to reduce blood pressure in SHR by acute i.v dosing of 3 mg/kg124. An aromatic ring directly attached to the 5-aminoindazole core was also present in ROCK-II inhibitor 45, where a quinazoline moiety functioned as the linker to connect the aminoindazole and a substituted phenyl group (Figure 4). Compound 45, also known as SLx-2119 or KD025, is the first reported specific inhibitor for ROCK-II. It has a high isoform selectivity with IC50 values of 60-110 nM for ROCK-II and > 10 µM125 or > 20 µM126 for ROCK-I. The quinazoline-(5amino)indazole 45 was first reported for ROCK inhibition in a Bayer patent application127 and was later reported as ROCK-II isoform selective inhibitors by Surface-Logix Inc126. Compound 45 has demonstrated to be effective for treating inflammatory and autoimmune disease128, cancer129, 130 and some CNS diseases131 in several relevant animal models. In addition to 5-aminoindazole, the 5-hydroxyindazole core can also function as an ideal scaffold for ROCK inhibition. As shown in Figure 4, compounds 46 and 47 by Bayer132 reported a 1,4-disubstituted phenyl group as a linker to attach the 5-hydroxyindazole core (the hinge binding moiety) to substituted pyrimidine groups. Highly potent ROCK inhibitors were obtained from this scaffold, as demonstrated by the IC50 values of 46 and 47 (20 nM and 6 nM for ROCKI, respectively). Interestingly, similar compounds derived from 4- and 6-hydroxyindazole also exhibited ROCK inhibition, but they were not as potent as the corresponding 5-hydroxyindazole counterpart132. UBE Industries reported two interesting series of ROCK inhibitors based on 5-substituted indazoles133, 134. Both series contained a terminal aminomethylene moiety, which was imported from 31 or 33 (Figure 3). The first series had an aromatic ring directly attached to the 5-position

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of indazole (via a C-C bond, structures not shown)133. The more interesting 5-substituted indazole based ROCK inhibitors from UBE is the second series released in a 2007 patent application, where a spiro side chain substituted aminomethlene moiety was directly connected to the 5position of the indazole core (48)134. Moreover, some compounds from this series could give a ROCK inhibition potency of IC50 < 10 nM134. Novartis recently published a novel class of ROCK inhibitors where an azaquinazoline ring was directly attached to 5-indazole (49, Figure 4)135. Potent and selective ROCK inhibitors were obtained from this class after chemical optimization. For example, compound 49 had an IC50 of 14 nM for ROCK-II, and was found to be selective against PKN and PKC135. In addition, inhibitor 49 demonstrated to be promising for the treatment of pulmonary hypertension (PH) in a mouse model by intra-tracheal dosing135. 3.3. 4-Carboxamido-aromatic ring substituted-pyridine (pyrimidine, pyrrolopyridine, etc.) based ROCK inhibitors. Compounds composed of an aromatic ring directly attached to the 4-position of a pyridine, pyrrolopyridine, or pyrimidine have long been used as scaffolds of ROCK inhibitors. The pyridine (or pyrrolopyridine or pyrimidine) moiety is supposed to be for hinge binding, and the terminal aromatic ring (see scaffold 50, Figure 5) is for binding towards the P-loop of the enzymes. GSK was the first to disclose this type of structures in its two PCT patent applications136, 137, where a carboxamide moiety was extended from the central aromatic ring (50). In the first application, a thiophene derivative was used as the central linker136 as shown in 51. More interesting ROCK inhibitors were disclosed in GSK’s second patent application, where a 4’-carboxamidophenyl group was attached to a hinge-binding pyridine or pyrimidine or pyrrolopyridine (52 to 54). In some structures, the amido nitrogen was ring-closured to the central phenyl ring (i.e. 54)137. No specific ROCK inhibition potency data were reported for this series from GSK. An interesting new development for this scaffold was disclosed recently by scientists

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from Bristol-Myers Squibb (BMS), where the tricyclic scaffold based inhibitor 55 (Figure 5) exhibited a potent ROCK inhibition with an IC50 of < 1 nM for ROCK-II138. 3.4. 4-Amido(urea or carbamate)-aromatic ring substituted-pyridine (pyrimidine, pyrrolopyridine, etc.) and phenoxy substituted-pyrrolopyridine based ROCK inhibitors. Instead of the 4’-carboxyl-phenyl moiety functioning as the central linker in 50, compounds containing a 4’-aminoaromatic ring-substituted pyridine/pyrimidine moiety (56, Figure 5) have also been widely used for ROCK inhibition. The amido-thiazole substituted pyridine 57 was reported to be a highly potent ROCK-II inhibitor (IC50 = 7 nM) by The Scripps Research Institute from their HTS campaigns32 but its optimization was discontinued mainly because of its high inhibition to CYP450 isoforms32. Similar ROCK inhibitors using a 5-membered heteroaryl moiety as the central aromatic ring have also been reported in several patent applications from Vertex Pharmaceuticals139-141. In Vertex’s ROCK inhibitors, the terminal amide was normally derived from a phenyl acetic acid derivative, such as compound 58 (Figure 5). Importantly, the 2-position of hinge binding pyridine in optimized inhibitors was generally substituted by an amino or a halo group55, 141

. This pyridine-2-substitution is supposed to be able to modulate the CYP450 inhibition55. For

example, compound 59, with a 2-fluorine-substituted pyridine and a terminal 3’(methansulfonamido)phenyl acetamide (Figure 5), was reported to have a ROCK-I inhibitory potency of Ki = 10 nM55. The crystal structure of 58 complexed with ROCK-I was discussed in section 2.2 and demonstrated that this class of ROCK inhibitors typically exhibited type-I kinase binding, with the pyridine moiety bound to the hinge region55. The H-bond between the methoxy (in 58) group and the backbone of F87 (or F103 in ROCK-II) explained why this class of compounds was selective against PKA55. Inhibitor 59 was profiled at 2 µM against a panel of 46 ki-

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nases, and found to inhibit only ROCK-I, ROCK-II, MSK1 and PRK2 with > 25% inhibition55. Compound 59 also demonstrated an excellent in vivo pharmacokinetic profile with an oral bioavailability of 69%, a clearance of 3.5 L/min/kg, and a half-life t1/2 = 2.6 h55. ROCK inhibitors based on 4-(4’-amido- or 4’-urea-phenyl) substituted pyridine were disclosed by both Astellas Pharma142 and the Moffitt Cancer Center54, 116. As shown in Figure 5, Astellas’ phenylalanine based amide 60 exhibited strong ROCK inhibition with IC50 = 5 nM. Compared to 60, the ROCK inhibitory activity of Astellas’ urea-based compound 61 (also coded as AS1892802143, Figure 5) was slightly less potent with an IC50 of 85 nM for ROCK-I. Interestingly, inhibitor 61 was selected by Astellas as a lead compound for further studies toward pain treatment143-146. This compound showed anti-nociceptive effects in adjuvant-induced arthritis (AIA) and monoiodoacetate-induced arthritis (MIA) rat models at an oral dosage of 0.1 mg/kg143, 145

, while at a dosage up to 3 mg/kg its blood pressure (BP) reduction effect on rats was

minimal145. Compound 61 was further tested in an osteoarthritis (OA) rat model, and an oral dosage of up to 10 mg/kg was required to generate significant effects146. The dosage required for OA applications (10 mg/kg) might create safety concerns because at this dosage 61 exhibited significant BP reduction145. BMS also applied the same tricyclic pyridine scaffold in 54 (Figure 5) to develop 4’-amidophenyl substituted pyridines as novel ROCK inhibitors147. Thus, the amide inhibitor 62 exhibited a high ROCK-II inhibition potency with a single digit IC50 value (6 nM). The carbamate 63 (Figure 5) showed a moderate ROCK-II inhibition (IC50 = 133 nM). It is speculated that substitution to its terminal benzyl moiety might be able to further enhance the potency, as was demonstrated by the SAR of other urea/carbamate based ROCK inhibitors52. Instead of the direct attachment (C-C) of an aromatic ring to the 4-position of a hinge binding pyridine, a novel class of ROCK inhibitors based on a 4-phenoxy substituted pyridine or pyr-

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rolopyridine scaffold (64, Figure 5) was disclosed by Bayer AG148. Compounds composed of the pyrrolopyridine hinge-binding moiety were discovered to be more potent than those containing a pyridine. Systematic optimization led to several highly potent ROCK inhibitors149. For example, compounds 65 to 67 were found to have IC50 values of 1-3 nM for ROCK-II149. Molecular docking studies of inhibitor 66 into ROCK-I showed that the pyrrolopyridine moiety bound to the hinge region with H-bonding to both E154 and M156, while its phenoxyaminopyrimidine part was pointing toward the catalytic loop of the enzyme149, 150. Large kinase panel profiling data were not available for this class but compound 65 was discovered to be selective against PKA and MLCK. Further, it had a good PK profile in rats with a clearance of Cl = 1.7 L/h/kg, a halflife of t1/2 = 1.2 h, and an oral bioavailability of 48%. In addition, at an oral dosage of 10 mg/kg, compound 65 induced significant BP reduction on both normotensive and spontaneously hypertensive rats150, and inhibited acute hypoxic pulmonary vasoconstriction (HPV) in isolated, ventilated and buffer-perfused murine lungs of rats151. 3.5. 4-Carboxamido-aromatic ring substituted pyrazole based ROCK inhibitors. ROCK inhibitors discussed so far all possess an ATP competitive kinase inhibition binding motif based on available X-ray structures. They use a pyridine, a pyrimidine, or a pyrrolopyridine, etc. as the hingebinding moiety. Recent discoveries from GSK, BMS, Roche, and The Scripps Research Institute revealed that the smaller 4-yl-pyrazole moiety can also function very well as the hinge binding moiety to develop ATP-competitive ROCK inhibitors. GSK disclosed the pyrazolephenylcarboxamide scaffold 68 (Figure 6) in the same patent application with their pyridinephenylcarboxamide scaffold 50 (Figure 5)137. No specific ROCK inhibition potency data were released in that application137. BMS also disclosed very potent ROCK inhibitors based on a similar scaffold (69, 70) in their recent patent application152. Compound 69 exhibited ROCK-I and

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ROCK-II inhibitory potency with IC50 values in the single-digit nanomolar range, while inhibitor 70 showed IC50 values in the sub-nanomolar range for ROCK-II152. 3.6. 4-Amido(ureido or carbamate)-aromatic ring substituted-pyrazole based ROCK inhibitors. In addition to the carboxamide phenylpyrazole-based ROCK inhibitor 68, BMS also disclosed its counterpart amido(ureido or carbamate)-based phenylpyrazoles (71) as ROCK inhibitors153. For example, compound 72 exhibited a high ROCK-II inhibition potency with an IC50 of 9 nM. In addition, it showed some isoform selectivity against ROCK-I (IC50 = 111 nM, ~11-fold isoform selectivity). Almost at the same time, Hoffman-La Roche revealed ROCK inhibitors also based on the amido-phenylpyrazole scaffold such as structure 73154, where a phenylpropanamide instead of the phenylacetamide in 72 was attached to the central aromatic ring (Figure 6). No biochemical ROCK potency was released for compound 73, but its cell-based potency in neurogenesis assays was reported to have an IC50 of 84 nM154. Several series of amidophenylpyrazole-based ROCK inhibitors were reported, with compounds 74 - 79 (Figure 6) as representative structures, by The Scripps Research Institute. The benzadioxane 3’-carboxamide SR3677 (74), which was developed from the optimization of the HTS lead 57 (Figure 5)32, had a high ROCK inhibitory potency in both biochemical (ROCK-II IC50: 3 nM) and cell-based (IC50: ~4 nM) assays32, 155, and was highly selective against its structurally close-related kinase PKA (IC50: ~ 4 µM)32. In addition, in the counter screen against a panel of 353 kinases (Ambit screen156-158), it was found to hit only 5 other kinases (Akt3, Clk1, Clk2, Clk4, and Lats2) with > 50% inhibition at 3 µM32. In the profiling against 70 non-kinase enzymes and receptors, 74 inhibited only three adrenergic receptors: α1a, α1b, and α2a (53%, 68%, and 76% inhibition, respectively, at 3 µM)32. 74 was also found to have low inhibition for the human-ether-a-go-go (hERG) at 3 µM (31% )32. More importantly, 74 was shown to facilitate

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aqueous humor outflow32, indicating its potential as an intraocular pressure (IOP) reducing agent. Inhibitor 74 also reduced BACE1 activity and amyloid-β production in an AD mouse model60, 159

. All these properties indicate that 74 is an excellent probe molecule for studying the biology

related to ROCK inhibition. The chroman-3’-carboxamide based ROCK inhibitor SR3850 (75, Figure 6)155,

160, 161

is

structurally a close analog of 74. It had a higher ROCK inhibitory potency than 74 with an IC50 of < 1 nM for ROCK-II, and better selectivity against PKA (IC50: > 20 µM) 160, 161. No profiling data was reported for 75 but its general kinase selectivity is expected to be similar to or even better than that of 74. Both 74 and 75 could therefore be used as probe molecules for ROCK inhibition. In addition, 75 had better in vivo PK properties than 7432 and was used for in vivo studies. In oral dosing at 2 mg/kg on rats, 75 exhibited an oral bioavailability of 35% and a half-life of 1 h (for a summary of PK for 75, see Table 1)160. In an acute oral dosing study, 75 reduced BP significantly (>20%) in both normotensive and SHRs at a dosage of 3 mg/kg with this effect lasting more than 10 hours (Feng, et al., unpublished results). In a study to determine the chiral preference for ROCK inhibition, the two enantiomers of 75 were each synthesized and evaluated, and the S-configuration was shown to be the more favorable enantiomer161. A series of amidophenylpyrazole based ROCK inhibitors derived from amino acids were also reported (76 to 79 in Figure 6)33, 162. The phenylalanine amide 76 presented a slightly lower potency than its counterpart pyridine-based amide 59 (Figure 5), while the phenylglycine amide 77 was a more potent ROCK inhibitor with an IC50 < 1 nM for ROCK-II. Compound 77 exhibited good cell potency in phosphorylation assays with an IC50 of 25 nM33. Systematic optimization from 76 and 77 led to inhibitor SR7043 (78)33, which had an IC50 in the sub-nanomolar range for

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ROCK-II and also exhibited some isoform selectivity against ROCK-I (IC50 = 10 nM). In addition, compound 78 showed an excellent selectivity against PKA (IC50 = 5130 nM). In a profiling study against a panel of 442 kinases (Ambit screen158), compound 78 hit only seven other kinases [Clk1(86%), Clk4 (91%), Lats2 (75%), MRCKβ (77%), NDR1 (72%), PI3Kβ (74%), and STK33 (66%)] at a concentration of 3 µM33. Since 3 µM is > 3000-fold of its ROCK-II IC50 value (< 1 nM), 78 is considered a highly selective ROCK-II inhibitor. The alkoxy side chain attached to the central phenyl ring in both 78 and 74 is believed the key structural element that contributes to the high kinase selectivity32, 33, 49, 163. Moreover, the application of this side chain can significantly increase the inhibitor’s aqueous solubility. Considering that it is also highly potent in cell-based phosphorylation assays (IC50 = 51 nM)33, 78 can be an excellent probe molecule for studying the biology related to ROCK-II inhibition. The IOP-lowering effect of 78 was evaluated in an elevated-IOP rat model164, 165 using Brown Norway rats. At an acute topical dosage of 40 µg, 78 significantly reduced IOP by > 20% and peaked at ~ 8 h33. The phenylpyrrolidine 3’-carboxamide 79162 was potent against ROCK-II with an IC50 value of 33 nM. Further optimization of this phenylpyrrolidine-based scaffold was not pursued mainly because of the presence of two chiral centers (Figure 6) in the molecule. Another interesting structure for ROCK inhibition is the mandelic acid derived compound 80162, which exhibited a high ROCK-II inhibition and a great selectivity against PKA. It has been proposed that the dimethylaminoethoxy side chain in 80 is responsible for its high selectivity. Urea/carbamate-derived phenylpyrazole ROCK inhibitors from SR644252 (81) to benzyl (4(1H-pyrazol-4-yl)phenyl)carbamate (85) have also been described52, 166-169. Inhibitor 81 is a direct analog of Astellas’ phenyl pyridine based ROCK inhibitor 61 (Figure 5). However, compound 81 had a much higher ROCK inhibitory potency (IC50 is 2 nM vs. 85 nM for 61) due to a

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4-yl-pyrazole (instead of the pyridine in 61) as the hinge binding moiety52. The move of the OHcontaining side chain from the benzyl α-carbon in 81 to the urea nitrogen atom resulted in compound 82, which had a slightly lower biochemical potency (IC50 = 12 nM) but had an improved cell-based potency in phosphorylation assays (IC50 is 59 nM vs. 150 nM for 81)52. The improvement in cell potency for 82 is likely due to a reduction of free NH/OH counts, which has been proposed to enhance the cell penetration capability170. Introduction of a methoxy group to the terminal phenyl ring of 82 on its 3’-position gave SR5834 (83)52, 166 that improved the biochemical ROCK-II potency (IC50 < 1 nM) and cell-based potency (IC50 = 19 nM), and enhanced the selectivity against PKA (IC50 = 3000 nM vs. 606 nM for 82)52, 166. These improvements might result from the methoxy group forming additional H-bonding with the backbone amide of residue F87 in ROCK-I or F103 in ROCK-II, as demonstrated by Vertex’s crystal structure for a similar ROCK inhibitor55. Both 82 and 83 had excellent in vitro and in vivo DMPK properties (see Table 1), which makes these compounds ideal candidates as probe molecules for in vivo studies. A dimethylaminoethoxy side chain is attached to the central phenyl ring in the un-substituted urea compound 84 (Figure 6). Like those in structures 74, 75, and 78, this tertiary aminecontaining side chain should enhance the inhibitor’s kinase selectivity. Indeed, 84 is very selective against PKA and a number of other kinases tested (such as JNK, p38, and MRCKα, in addition to PKA)166. Moreover, 84 showed an excellent cell-based potency with an IC50 value of < 4 nM in phosphorylation assays166. Inhibitor 84 was also tested in an elevated-IOP rat model164. At a topical dosage of 40 µg, 84 reduced IOP by > 20%, with the effect lasting for > 4 h166. Finally, in addition to compounds derived from urea, carbamate-based phenylpyrazole compounds also demonstrated good ROCK inhibition. For example, compound 85 exhibited an IC50 of 7 nM for

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ROCK-II. Even without any substitutions to its terminal and/or central phenyl rings, 85 showed a great selectivity against PKA (IC50 > 20 µM)167. 3.7. 4’-Alkylaromatic ring substituted pyrazoles as ROCK inhibitors. In addition to 4’carboxamide (68) and 4’-amido/ureido/carbamate (71) substituted phenylpyrazoles, Astex Pharmaceutics disclosed a series of 4-alkyl-substituted phenylpyrazole compounds as ROCK inhibitors (86, Figure 7)171-173. In these structures, an sp3-carbon is directly connected to the central phenyl group or other aromatic rings. The benzyl-type α-carbon (or sp3-carbon) was substituted both by a group containing an aromatic moiety and by a second alkyl chain normally substituted by at least one heteroatom such as a nitrogen or oxygen atom. For example, a chlorophenyl moiety was present in all structures 87 to 91, and an N-containing alkyl substitution was also directly attached to the sp3-carbon of the 4’-yl-phenylpyrazole in these compounds (Figure 7). No specific ROCK potency data were reported in the Astex patent applications but the IC50 values of compounds 87 to 91 were reported to be all < 100 nM171. Interestingly, the chirality of the sp3-carbon did not have much influence on the ROCK inhibitory activity for 88 and 89. General kinase selectivity data were not available for these ROCK inhibitors, however, they inhibited p70S6K, PKA, PKB etc.172, 174-178. Further modifications of this scaffold are necessary in order to obtain highly potent ROCK inhibitors with reduced PKA inhibitory activity and improved general kinase selectivity. 3.8. Benzimidazole, benzothiazole, indazole, and indole substituted pyrazole derivatives as ROCK inhibitors. The pyrazole based ROCK inhibitors discussed above in Figures 6 and 7 are all based on a mono-cyclic aromatic ring as the central structural element. Several classes of pyrazole-based ROCK inhibitors using a bis-cyclic aromatic ring ([6,5]- and [6,6]-fused ring) as the

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central moiety were also disclosed by the Scripps Research Institute in their patent applications179-182 and publications49-51, 163, 183-185. In these structures, the fused ring was normally a 5- or 6-membered heterocyclic ring. Representative ROCK inhibitors for these scaffolds based on [6,5]-fused rings are shown in Figure 8. When a benzylamine based carboxamide is attached to the 2-position of the central benzimidazole ring, highly potent and selective ROCK inhibitors could be obtained. For example, compound 92 (Figure 8) exhibited an IC50 of 4 nM for ROCK-II179. This molecule was highly selective against PKA (~ 380-fold selectivity) even though it did not possess a side chain substitution on its central benzimidazole ring. This is unique, as most carboxamide and urea derived compounds based on scaffolds 68 and 71 did not show good PKA selectivity when side chain substituents (such as those in 74, 75, 76, and 84) were absent on their central phenyl rings. Apart from the location of the terminal benzyl moiety, the structural skeleton of 92 is similar to those in 71 and 81−84. Interestingly, when a 3’-yl-chromane ring was directly attached to the 2-position of the benzimidazole core (compound 93), slightly lower ROCK potency and lower PKA selectivity were obtained (Figure 8)51. However, when the hinge binding pyrazole was replaced by an aminopyrimidine moiety to give compound 94 (Figure 8)51, high ROCK inhibitory potency and high PKA selectivity (> 480-fold) were obtained. The comparison of both ROCK inhibition potency and PKA selectivity among compounds 92−94 indicated that the distance (and maybe the orientation) between the hinge-binding moiety and the terminal aromatic ring was critical for an optimal binding to the ATP pocket of ROCK enzymes. The combination of pyrazole and benzylamine-carboxamide (92) and the combination of aminopyrimidine and 3’-yl-chromane (94) are both optimal, while the combination of pyrazole and 3’-yl-chromane (93) is only sub-optimal for this benzimidazole-based scaffold.

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A different result was obtained when a benzothiazole was used as the central moiety. While potent and PKA-selective ROCK inhibitors could still be obtained from the pyrazole and benzylamine-carboxamide combination, as demonstrated by compound 95 (Figure 8)184, only moderate ROCK inhibition was found from compounds based on the aminopyrimidine and 3’-yl-chromane combination (96, Figure 8). It is speculated that the benzothiazole ring is larger than the corresponding benzimidazole ring and that consequently the aminopyrimidine and 3’-yl-chromane combination in 96 had affected the optimal hydrophobic interactions between the chromane aromatic moiety and the hydrophobic pocket under the P-loop which are present in inhibitor 94. The structural skeleton of ROCK inhibitors based on a 3-carboxamide-6-pyrazole indazole scaffold (compounds 97 to 100, Figure 8) is very similar to that of the urea and carbamate-based ROCK inhibitors (71, 81 to 85), where the NH group in urea or carbamate compounds is replaced by a C=N moiety in indazoles. Therefore, it is anticipated that the resulting molecules will be highly potent49, 179, 183. The IC50 values of compounds 97−100 for ROCK-II were all in the single-digit nanomolar to sub-nanomolar range (Figure 8). Similar to compound 92 and unlike the urea-based scaffold 71, high selectivity against PKA could be obtained even without attaching a tertiary amine-containing side chain to the central aromatic ring (97−99). Interestingly, when a dimethylaminomethyl side chain was attached to the amide nitrogen (100), the PKA inhibitory activity was enhanced (Figure 8). Moreover, when the 4’-yl-pyrazole was attached to the 5-position (instead of the 6-position in 97) of the central indazole ring (101), ROCK inhibition was significantly reduced (101, IC50 = 67 nM vs. < 1 nM for 97, Figure 8). Similar results were also obtained in the indole series of ROCK inhibitors49, 179, 183. Replacement of the 2-nitrogen in the indazole by a carbon atom does not affect the ROCK affinity. The

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3-carboxamide compound 102 is a direct analog of the indazole based 97, and demonstrated a greater ROCK-II inhibition with an IC50 < 1 nM (Figure 8). The move of the carboxamide from 3-position to 2-position yielded compound 103, which is a direct analog of the benzimidazolebased inhibitor 92. Again, inhibitor 103 was highly potent against ROCK-II with an IC50 of 1.6 nM (Figure 8). Like 92 and 97, both 102 and 103 had high selectivity against PKA (> 3000-fold) even though they had no functional groups substituted on their central indole ring. Conversely, although a dimethylaminomethyl substitution on the indole 3-position of 103 generated a molecule which still exhibited strong ROCK inhibition (104, IC50 = 9 nM), the selectivity over PKA for this new inhibitor 104 was slightly reduced (~1600-fold for 104 vs. > 3000-fold for 92 and 97). When the pyrazole was attached to the 5-position of the central indole moiety (rather than the 6-position in 102−104), a reduction of ROCK inhibitory potency was observed for the 3carboxamide based compounds. For example, the IC50 of the 5-substituted compound 105 (Figure 8) was 67 nM, which was significantly higher than that of its 6-substituted counterpart 102 (< 1 nM, a greater than 67-fold reduction in potency). Interestingly, for the 2-carboxamide indole series (103 and 104), the reduction of ROCK potency was not so significant when moving the pyrazole from 6-position to 5-position of its central indole ring (compound 106, IC50 = 17 nM vs. 1.6 nM for 103, ~ 10-fold reduction of ROCK potency). This may be due to the fact that, in the 2-carboxamide series, the move of pyrazole from 6-position to 5-position did not significantly change the conformation/orientation of the molecule, and thus would not affect the binding affinity as much as that in the 3-carboxamide series. 3.9. Quinazolinone substituted pyrazole derivatives as ROCK inhibitors. In addition to [6,5]fused rings, ROCK inhibitors were also reported based on a [6,6]-fused quinazolinone moiety as the central aromatic ring163, 182, 185. In this series of ROCK inhibitors, an alkyl group, which nor-

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mally contains a terminal aromatic moiety, is directly attached to the central quinazolinone ring on its 2-position163, 182, 185. For example, in compound 107 (Figure 9), a 2’-yl-benzadioxane was attached to the 2-position and the pyrazole was substituted on the 6-position of a quinazolinone core. This compound exhibited strong inhibition to both ROCK-I and ROCK-II at an IC50 of 1 nM, but it also potently inhibited PKA (IC50 = 2 nM)163. Replacement of the benzadioxane in 107 with a 6’-methoxy-3’-yl-chroman group, yielding SR8046 (108, Figure 9)163, resulted in a molecule which not only retained potent ROCK inhibition, but also led to a significant enhancement of PKA selectivity (ROCK-II/PKA selectivity for 108 is > 140-fold vs. 2-fold for 107)163, 182

. The inhibitory activity against PKA can be further decreased by introducing an alkoxy sub-

stitution on the 8-position of the quinazolinone core, as demonstrated by SR6246 (109, PKA IC50 > 14 µM) and 110 (PKA IC50 > 20 µM)163. The larger the side chain, the lower the PKA inhibition (109 vs. 110). The long chain alkoxy substituent can also be applied to the 6’-position of the chroman ring (111, Figure 9) to provide potent and selective ROCK inhibitors. Interestingly, the switch of the methoxy and dimethylaminoethoxy groups between the quinazolinone 8-position and the chromane 6’-position did not change the inhibition profile. Compounds 110 and 111 had a similar inhibitory profile against ROCK-I, ROCK-II, and PKA. In addition, the dimethylaminoethoxy side chain (in 110 and 111) is expected to improve the aqueous solubility significantly. Importantly, the introduction of a dimethylaminoethoxy side chain, either on the quinazolinone 8-position or at the chroman 6’-position, induced some isoform selectivity favoring ROCK-II inhibition (Figure 9). Both compounds 110 and 111 exhibited a ~ 10- to 12-fold isoform selectivity of ROCK-II over ROCK-I, which was higher than that of compound 109 (~ 4-fold). Molecular docking studies for the quinazolinone scaffold indicated that the substituent on the quinazolinone 8-position bound to the catalytic loop and to the solvents, while the substitution group on

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the 6’-position of the chroman ring was immersed under the P-loop163, 185. Therefore, exploring these two areas of ROCK-I and ROCK-II might provide important insights for designing isoform selective ROCK inhibitors. In addition to the benzadioxane and chroman moieties, substituents composed of structural elements from amino acids were also evaluated for ROCK inhibition182, 185. Thus in compounds 112−114 (Figure 9), structural elements from a phenylalanine (112) or a phenylglycine (113, 114) were attached to the 2-position of the 6-pyrazolequinazolinone core. Compared to their benzadioxane and/or chroman-based counterparts, these amino acid-based compounds had a similar or slightly less potent ROCK inhibition. However, no significant PKA selectivity, even with an 8-methoxy substitution (112 and 114), could be achieved. These compounds might be useful probe molecules for ROCK/PKA dual inhibition185. Replacing the 6-position pyrazole by a pyridine (115) or an aminopyrimidine (116) as hingebinding moieties resulted in significant reduction in ROCK inhibition potency (Figure 9). For example, compound 115 is > 50-fold less potent than its counterpart 107, and compound 116 has > 130-fold reduced potency compared with the corresponding inhibitor 108. These results demonstrated that 4’-yl-pyrazole is a preferred hinge-binding moiety over a 4’-yl-pyridine or 4’yl-aminopyrimidine for the quinazolinone core. In addition, the shift of pyrazole from 6-position to 7-position of the quinazolinone also significantly reduced the ROCK inhibition (117 vs. 108, Figure 9). Surprisingly, a methyl substitution to the quinazolinone amide NH (118, Figure 9) led to > 2300-fold decrease in ROCK inhibition potency (118 vs 108), indicating that either the amide NH is functioning as an H-bond donor or there is no room to accommodate a methyl group in the ATP binding pocket.

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Like the pyrazole-based ROCK inhibitors derived from chroman amide and urea, ideal in vitro and in vivo DMPK properties were also obtained for ROCK inhibitors based on the quinazolinone scaffold (107 – 111, Table 1). Much higher stability was observed in human microsomes than in rat microsomes. These compounds also demonstrated a good CYP450 inhibition profile except that of 107, 110, and 111 for the 2C9 isoform. 108 and 109 exhibited excellent properties for i.v dosing, with high Cmax and AUC, and low clearance and low volume of distribution. The very low oral bioavailability for 110 and 111 may result from their low microsomal stability on rats and high plasma clearance. Compounds in Table 1 might be good probe molecules for in vivo studies. 3.10. Benzathiophene substituted pyridine (pyrimidine, pyrrolopyridine) derivatives as ROCK inhibitors. ROCK inhibitors presented in Figure 8 are all based on [6,5]-fused rings as the central aromatic ring. Kalypsis reported a class of ROCK inhibitors that used a [5,6]-fused ring as the central aromatic moiety186. In structures 119 to 121 (Figure 10)186, 187, the hinge-binding aminopyrimidine is attached to the 2-position of a thiophene core, which was used as the central aromatic ring. The phenyl ring of the benzathiophene was either un-substituted, as in 119, or was substituted on its 5- or 6-position by a functional group, such as an alkyl group, an amido, a carboxamide, or a urea186. Strong ROCK inhibition was observed for these benzathiophene-based ROCK inhibitors. For example, compound 121 showed an IC50 of 3 nM for ROCK-II187. The IOP-lowering effect of 121 in monkeys was also evaluated by topical ocular dosing. At a dose of 250 µg, a 26% reduction in IOP was observed at 6 h post administration187.

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3.11. Benzamide derivatives as ROCK inhibitors. Benzamide derivatives have been described as ROCK inhibitors in several patent applications. Sumitoma Pharma first disclosed a primary benzamide based scaffold in the early 2000s188. Compound 122 (Figure 10), a representative inhibitor from this scaffold, had ROCK inhibitory activity of IC50 = 26 nM188. No further biological evaluation of this series of ROCK inhibitors has been described, but the primary amide is thought to be hinge-binding and these compounds are typical type-I kinase inhibitors. Boehringer Ingelheim described a secondary benzamide based scaffold in its 2008 patent application189. Compounds reported in this application exhibited strong ROCK inhibition as demonstrated by structure 123 (Figure 10) with an IC50 of 6 nM for ROCK-II188. No further studies were reported by Boehringer Ingelheim for this series. The binding mode of this class of ROCK inhibitors is not clear but it is likely that one of the two benzamide moieties would be a hinge binder if the compound binds to the ATP pocket. 3.12. Aminofurazan derivatives as ROCK inhibitors. GSK reported an interesting series of ATP competitive ROCK inhibitors that used an aminofurazan as the hinge binding moiety190, 191. The scaffold structure 124 (Figure 10) was docked into ROCK-I in order to provide insights for SAR optimization. In addition to hinge-binding from the aminofurazan, the pyridine nitrogen from the central azabenzimidazole ring was found to form an H-bond with the side chain of catalytic residue K105, which is supposed to be a key factor that contributed to the high potency of 124 (IC50 = 19 nM)190. Systematic optimization from 124 led to highly potent ROCK inhibitors that had much improved selectivity against ASK1 and several other kinases, and also had excellent in vivo pharmacokinetic properties. For example, both compounds 125 and 126 (Figure 10) had a sub-nanomolar IC50 value against ROCK-I as well as good oral bioavailability of 40% and 69%, respectively190. The anilinobenzamide group in 125 and 126 is thought to bind to the P-loop, and

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this additional hydrophobic interaction is responsible for improved affinity. Significant BP reduction was observed for 126 at an oral dosage of 3 mg/kg in SHR190, 191. No profiling data against a large panel of kinases has been reported for this series of ROCK inhibitors. 3.13. 6-/7-Substituted isoquinoline/isoquinolinone derivatives as ROCK inhibitors. Isoquinolines have long been for the basis of multiple ROCK inhibitors, where the isoquinoline core is functioning as a hinge-binding moiety. In addition to the 5-substituted isoquinoline/isoquinolinone (which was the basis of the first scaffold used in the classical ROCK inhibitor 28 and derivatives, those described by Kirin111-113 and Inspire Pharmaceuticals114, 115, 192), 6- and 7-substituted isoquinoline derivatives were also revealed to have high ROCK inhibition by Aerie Pharmaceuticals and Sanofi-Aventis. The 6-amido-isoquinoline scaffold 127 (Figure 11) was reported by Aerie193. In this scaffold, an amino moiety is normally present in R group and an aromatic moiety always exists in ring A. No specific ROCK inhibition potency data were available from compounds in this series but several lead inhibitors are currently in or close to clinical trials for treatment of ocular hypertension and glaucoma194-196. More details will be discussed in Section 4. Instead of a 6-amido-substitution, ROCK inhibitors based on structures with a saturated ring attached to the 6- or 7-position of the isoquinoline/isoquinolinone core through a –S-, or –O-, or –NH- linkage were described by Sanofi-Aventis

197-200

. The saturated ring B in 128 and 129

(Figure 11) could be a heterocycle or a simple carbocyclic ring201. If it is a simple carbocyclic ring, it is generally substituted by a group containing an amino moiety201. No SAR analysis was described for this series of ROCK inhibitors but one lead compound, SAR407899 (171, Figure 16), was reported to have a Ki of 36 nM for ROCK-II202. Compound 171 was profiled at 10 µM against a panel of 93 kinases and was found to hit 4 kinases with IC50 of a few µM. In a

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screening against 117 non-kinase enzymes, it inhibited only moderately µ-opioid and serotonin transporter, again with IC50 of a few µM202. 171 was demonstrated to be effective for the treatment of hypertension202-204 and erectile dysfunction (ED)205, and was in clinical trials by SanofiAventis. More details will be discussed in Section 4. 3.14. Boron derivatives as ROCK inhibitors. Almost all ATP competitive ROCK inhibitors reported to date utilize a heteroaryl ring containing at least one nitrogen atom as the hinge binding moiety. Nonetheless, Anacor Pharmaceuticals recently published a novel class of ROCK inhibitors that used a benzoxaborole as a hinge binding moiety56, 206. As shown in Figure 12, scaffold 130 also has a phenoxy-substitution on its 6-position in addition to the hinge-binding benzoxaborole56. The discovery of ROCK inhibitors from this class of compounds was based on unexpected properties (i.e. inhibition of Toll-like receptor–stimulated cytokine secretion from leukocytes) exhibited by benzoxaborole-based drugs under development at Anacor56. Profiling against a large panel of kinases (Ambit screen157) demonstrated that this type of benzoxaborole-based compound inhibited ROCK, PKA, p38, and a number of other kinases. The inhibition potency of lead compounds (131−133) for ROCK was moderate with IC50 values in the range of a few hundred nM (Figure 12)56. The co-crystal structure of human ROCK-II complexed with 131 revealed that the benzoxaborole head (oxaborole) was indeed bound to the hinge with H-bond formation between the OH group and E170 and between the oxygen atom and M172. In addition, the aminomethyl group of compound 131 occupied the magnesium cofactor site, and formed H-bonds with both N219 and D23256. SAR studies showed that the boron atom was indispensable and the ROCK inhibition was totally lost when boron was replaced by a carbon atom56. It is important to note that the selectivity

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of these benzoxaborole inhibitors against PKA and a number of other kinases is moderate with the best PKA selectivity being only ~ 15-fold among those tested56. Compound 131 had a poor in vivo PK profile with high clearance and low oral bioavailability. Its chlorine-substituted counterpart 133 had improved PK properties with a clearance of 10 L/kg, and an oral bioavailability of 45% in the saline/PEG400/DMSO solution (55:35:10) and 107% in the water-cyclodextrin formulation (84:16)56. At an oral dosage of 600 mg/kg, inhibitor 133 significantly lowered the BP of SHRs and normotensive rats56. The high dose (i.e. 600 mg/kg) required of compound 133 is largely due to its moderate ROCK inhibition activity and high systemic clearance. Optimization of lead compounds by Anacor led to more potent compounds. For example, compound 134 showed a ROCK inhibition IC50 of ~ 50 nM206. However, no results of further biological studies for 134 were disclosed. Interestingly, this kind of benzoxaborole-based compound (135, Figure 12) has also been used to develop β-lactamase inhibitors207. 3.15. Development of ROCK inhibitors for localized applications. The discussion of ROCK inhibitors in Section 3 thus far has been mainly based on different classes of hinge-binding moieties. However, optimization of pharmacokinetic properties to create candidates with reduced systemic exposure is another recent and interesting development in the field of ROCK inhibition. Indeed, the therapeutic window of systemically available ROCK inhibitors is narrow mainly because of the pronounced BP reduction and the associated increase in heart rate (HR) 56, 150, 203-205, 208, 209

. In addition, systemic ROCK inhibition was found to induce reversible reduction in lym-

phocyte counts135. The development of ROCK inhibitors for acute applications and/or for conditions that can be treated by localized drug action therefore represents an attractive alternative approach210, 211. A soft drug approach211-222 has been applied by scientists at Amakem NV to develop ROCK inhibitors for localized applications such as the treatment of glaucoma, age-related

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macular degeneration (AMD) and chronic obstructive pulmonary disease (COPD). In this approach, ROCK inhibitors are delivered locally to the targeted organs. Once they enter the systemic circulation, these ROCK inhibitors are metabolized into nontoxic and/or inactive metabolites217. Several patent applications have been filed by Amakem based on these “soft” ROCK inhibitors223-229. The strategy applied by Amakem was to add an ester moiety to an otherwise classical ROCK inhibitor in such a way that it did not reduce or potentially could enhance the ROCK affinity. Molecular variations on the resulting ROCK inhibitors aimed to combine high stability in target organs with rapid inactivation by various esterases in the systemic circulation (t1/2 in plasma is < 5 min)215, 223, 224. Soft ROCK inhibitors developed to date are all based on classical scaffolds. For example, the 5-aminoisoquinoline scaffold of ROCK inhibitors 136 (Figure 13) was originally developed by Kirin111, 112, and modified by Amakem through the addition of an ester moiety to the 3’-position of the terminal benzylamine to make the compound soft-drug-like223. The 5aminoindazole scaffold was also identified by Kirin111, 113, and was adapted by Amakem via the insertion of an ester group to the terminal benzylamine moiety to give a highly potent soft ROCK inhibitor 137 (Figure 13)223. Plasma stability could be manipulated by varying the ester moiety. Particularly, addition of a methyl ester to 136 and 137 yielded a t1/2 > 30 min223, while the tetrahydrofuran-2-yl-methyl ester 138 had a t1/2 < 5 min in plasma223. In addition, after the ester modification, compound 138 still retained a high ROCK-II potency with an IC50 of 3 nM. Other interesting soft ROCK inhibitors were derived from compound 33 (Figure 3). As shown in scaffold 139 (Figure 13), an additional phenyl ring was found to be tolerated on the 3’position of the central benzamide ring of the 33 scaffold230-232. A meta-ester substitution on this

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new aromatic ring was demonstrated to yield highly potent soft ROCK inhibitors217. For example, all three esters 140 to 142 had ROCK-II IC50 values in the single-digit nanomolar range. In addition, they also exhibited good cell potency (Figure 13). The docking mode of an analog of scaffold 140 in ROCK-I showed that, in addition to the hinge binding of pyridine, the new aromatic ester moiety bound to an area under the P-loop and the ester was H-bonded to F87217. The variation in human plasma stabilities for these three esters demonstrated the possibility of manipulating compound stability in plasma (and in targeted organs, data not shown). For instance, the methyl ester 140 had a t1/2 = 65 min, while the propyl ester 141 gave a lower stability of t1/2 = 19 min. The tetrahydrofuran-2-yl-methyl ester 142 had the lowest stability with a t1/2 < 5 min, which will obviously reduce systemic exposure217. Molecular modeling studies of an analog of 140 indicated that another aromatic ring attached to 140 via an amide linkage might further improve the inhibitor’s potency because of extra πstacking interactions from this additional aromatic ring with the side chain of F120 in ROCK-I or F136 in ROCK-II57. As with earlier derivatives, an ester moiety was linked to this new aromatic ring as shown in structures 143 to 150 (Figure 13). ROCK-II inhibition data for these compounds demonstrated that they were indeed more potent than 140 to 142. Interestingly, molecular variations in this series indicated that plasma stability could be modulated not only by changing the alcohol chain, but also by modifying other parts of the molecule. For 3’-benzolyl esters, this is illustrated not only by the lower stability of 143 compared to 140, but also by the differences in plasma stability that can be obtained through simple fluorine substitution (144) or reversal of an amide linker (145). In such cases, plasma stability can still be modulated through variation of the alcohol chain, as demonstrated by 146. On the other hand, 4’-yl-esters appear much more stable in plasma (147, 148) and had t1/2 > 120 min (Figure 13), although rapid ester hydrolysis in pres-

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ence of hepatocytes was demonstrated for 147. Replacement of the benzoyl ester by a phenyl acetyl ester (149 and 150) resulted in distinct structure-stability relationships, as both 3’-yl and 4’-yl derivatives displayed plasma t1/2 < 5 min. The crystal structure of 144 complexed with ROCK-II validated the predicted binding modes of this compound series57. In vivo studies demonstrated the therapeutic potentials of Amakem’s soft ROCK inhibitors. Compound 142 was shown to lower IOP in normotensive rabbit eyes following topical administration217. AMA0076 (structure not disclosed) potently lowered IOP in New Zealand white rabbit eyes with minimal hyperemia233, indicating that soft ROCK inhibitors can indeed minimize the major side effect associated with systemic ROCK inhibition234. Another soft ROCK inhibitor, AMA0428 (structure not disclosed), was shown, in a dose-dependent manner, to not only reduce neoangiogenesis, but also block inflammation and fibrosis, suggesting a potential therapeutic benefit of ROCK inhibition in neovascular (or wet) age-related macular degeneration (AMD)235. Importantly, oral dosing of Amakem’s soft ROCK inhibitors at a dosage suitable for localized drug application did not lead to obvious BP reduction, further indicating the therapeutic potential of soft ROCK inhibitors (Defert, et al., unpublished results). Development of AMA0076 has advanced into the clinic and the molecule will be discussed in greater detail in Section 4. 3.16. ROCK-II isoform selective inhibitors. Another important direction for future ROCK inhibitor development is the discovery of ROCK-II specific inhibitors210. Until very recently, the aminoindazole-quinazoline based 45 (Figure 4) was the only reported ROCK-II specific compound. After Kadmon Corporation acquired the intellectual property of 45, it expanded this quinazolinebased scaffold by replacing the central quinazoline with a monocyclic heteroaryl ring236. When the central heteroaryl ring is substituted by an isoindoline or tetrahydroisoquinoline through a C-

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N linkage, as shown in structure 151 (Figure 14), very potent and isoform selective ROCK-II inhibitors could be obtained. For example compounds 152 and 153, which used a 5aminoindazole as the hinge-binding moiety and had a terminal isoindoline group attached to the 2-position of the central pyrimidine core, were both highly potent ROCK-II inhibitors (IC50 was 10 nM and 50 nM, respectively) and had high isoform selectivity against ROCK-I (selectivity was > 89-fold and > 150-fold, respectively). More interestingly, 4-yl-phenylprazole and 4-ylphenylpyridine could also be used as the hinge-binders to produce potent and isoform selective ROCK-II inhibitors. More specifically, the pyrazole based compound 154 had a ROCK-II IC50 of 2 nM and the pyridine based compound 155 exhibited a ROCK-II inhibition with IC50 of 35 nM, while both inhibitors had low activity against ROCK-I with IC50 > 10,000 nM. All four inhibitors in Figure 14 (152−155) showed good cell potency in phosphorylation assays against the myosin light chain (MLC) substrate236. No kinase and/or non-kinase profiling data were released for these isoform selective ROCK-II inhibitors disclosed in the recent Kadmon patent application236, and therefore there is uncertainty about their general selectivity other than their stated high isoform selectivity of ROCK-II over ROCK-I. 3.17. Bis-functional ROCK inhibitors. Another recent evolution in the field of ROCK inhibitors is the discovery and development of bis-functional small molecule inhibitors which comprise both ROCK inhibition plus another therapeutically relevant non-kinase target. An example for this trend is Abbott’s ROCK-NAMPT (nicotinamide phosphoribosyltransferase, also known as pre-B-cell colony-enhancing factor 1) dual inhibitors. Inhibition of NAMPT has therapeutic potential for cancer237-239, inflammatory diseases and metabolic disorders239, 240. Given the potential therapeutic applications of ROCK inhibition, the simultaneous inhibition of both ROCK and

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NAMPT could have additive or even synergistic effects. As shown in structure 156 (Figure 15), Abbott disclosed a class of 4-phenyl substituted phthalazinone or isoquinolinone-based structures that showed potent inhibition of both ROCK and NAMPT241. Compounds 157 – 159 (Figure 15) are some examples of Abbott ROCK-NAMPT dual inhibitors. Interestingly, BMS disclosed that similar phthalazinone or isoquinolinone-based structures, following replacement of the terminal ureido-isoindoline in 156 by a variety of other moieties (160), still showed pan-ROCK inhibition242. Structures 161 – 164 (Figure 15) are representatives for the series. No binding information was revealed for these compounds but it is speculated that they are ATP-competitive ROCK inhibitors with the amide from the phthalazinone or isoquinolinone core bound to the hinge region. No specific potency data were disclosed for this series of ROCK inhibitors, but many compounds were reported to have an IC50 < 100 nM for ROCK-II242. In addition, it is not clear whether these BMS compounds also have NAMPT inhibitory activity. Other than biochemical data, no cell potency data, DMPK properties or other profiling data are available for either Abbott’s ROCK-NAMPT dual inhibitors or BMS’s pan-ROCK inhibitors. More recent developments in bis-functional ROCK inhibitors include a publication by Alokam et al. in early 2015 describing dual inhibitors of ROCK-I and NOX2 (NADPH oxidase) for the treatment of progressive neurological diseases such as AD, autism spectral disorder, and fragile X syndrome243. In 2014, Chen et al. described small molecules created by the incorporation of lipoic acid into a ROCK inhibitor (28) with potential therapeutic application for the treatment of CNS diseases244. In these compounds, the lipoic acid moiety was covalently connected to a ROCK inhibitor, and the resulting inhibitors were bis-functional both in vitro and in vivo. Inspire Pharmaceuticals reported a series of bis-functional ROCK inhibitors for treatment

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of glaucoma 192, 245. Since the 1990s, prostaglandin derivatives have been used for reducing IOP in glaucoma patients246, 247, thus the simultaneous application of a ROCK inhibitor and a prostaglandin derivative might have additive or even synergistic effects. The Inspire dual inhibitors were designed and developed by combining a ROCK inhibitor and a prostaglandin derivative via a covalent linkage. Unlike the lipoic acid-ROCK dual inhibitors244, the covalent linkage in the Inspire compounds was usually an ester (such as a carbamoylmethyl ester) and could easily undergo metabolism to release the original ROCK inhibitor and the prostaglandin derivative in vivo192, 245. Therefore, the Inspire dual inhibitors are bis-functional only in vitro. 3.18. Appropriate probe molecules for ROCK inhibition? As summarized above, the past 20 years have witnessed major advances in the development of potent and highly selective ROCK inhibitors. However, it is interesting to note that, almost exclusively, the biology world still utilizes the classic ROCK inhibitors, 28 and 31, for probing the Rho pathways, and that these molecules are still considered to be the “gold standard” for pharmacological ROCK inhibition. This is probably mainly due to the fact that these two classic ROCK inhibitors are commercially available and their structures are simple. However, both 28 and 31 have low ROCK inhibition and poor cell potency (biochemical IC50 is > 100 nM for 31 and > 300 nM for 28 reported in most publications, and their cell based IC50 values are in the micromolar range) and additionally they are unselective against a range of other kinases, especially those in the AGC family93, 248. When a high concentration of 28 and /or 31 is used in probing studies (especially in vivo studies), it is unclear whether the obtained results are due to on-target or off-target effects. In light of the fact that there are so many newly developed, potent and selective ROCK inhibitors which have been disclosed over the past few years, it would be more logical and appropriate to utilize one of these new ROCK inhibitors as alternative probes for biological studies.

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Many compounds reviewed in this Perspective could serve as better ROCK probe molecules than 28 and 31. For example, compounds 43, 45, 46, 61, 65, 74, 75, 78, 83, 108, 109, 126 and 154 are all good candidates for both in vitro and in vivo probing studies, as these molecules are much more potent and more selective than either 28 or 31. The availability of pharmacokinetic data for many of these molecules is helpful when planning in vivo studies. In addition, profiling data against a large kinase panel (74 and 78) and non-kinase enzymes (74) are available for prediction of potential off-target effects. Even without profiling data, the kinase selectivity for the candidate ROCK inhibitors listed above is expected to be better than that of 28 or 31 based on available SAR data and structures. When evaluating ROCK-II specific inhibition, either 45 or 154 might be the optimal choice since both inhibitors have an isoform selectivity of > 100-fold. Some isoform selectivity (> 10-fold) is also found in 72, 74, 78, 110, and 111. Data resulting from the evaluation of 147 in a broad kinase selectivity panel (Reaction Biology Corporation) is also available57. 4. Current Status of ROCK Inhibitors Despite the significant interest from major pharmaceutical and small biotech companies in the ROCK pathway and the pleiotropic effects of ROCK inhibitors, there are relatively few ROCK inhibitors that have either reached the stage of clinical trials or even the market. Inhibitor 28 has been marketed for treatment of cerebral vasospasm since 1995 in Japan and has also been approved in China. Recently, 165249 has also been approved for the treatment of glaucoma in Japan. In this section we will review the status of ROCK inhibitors currently in clinical trials or close to the clinic for various therapeutic indications including ocular, respiratory, dermal diseases, erectile dysfunction, and cancer.

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4.1. ROCK inhibitors for the treatment of glaucoma. In 2010, more than 60 million people worldwide were affected by glaucoma and this number is expected to increase to 80 million by 2020250. The most common form of glaucoma is primary open-angle glaucoma (POAG) and it is believed that almost 1% of the American population have this form of glaucoma (https://www.glaucomafoundation.org). Although the etiology of glaucoma is not yet fully understood, it is widely accepted that elevated IOP is so far the only treatable risk factor251. While there are numerous classes of drugs approved for IOP reduction in patients with glaucoma, there remains an unmet need for novel drugs that can lower IOP with improved safety/tolerability. In particular, because no commonly used drugs target the main outflow channel (i.e. trabecular outflow), drugs such as ROCK inhibitors that do target this pathway are needed. As described previously, the development of ROCK inhibitors has been severely hampered by unwanted cardiovascular side effects, resulting in a very narrow therapeutic window. Topical ocular administration is usually seen as a safe approach to limit or avoid systemic exposure to drugs. Given the mechanism of action of ROCK inhibitors86, significant efforts have been made in their development for the management of elevated IOP in glaucoma. Consequently, over the past few years, several ROCK inhibitors have been evaluated in clinical trials (Table 2)252, 253. However, the use of ROCK inhibitors for the treatment of eye diseases is still limited by conjunctival hyperemia (redness of the eye), which is caused by vasodilation of the conjunctival vasculature. Recently, compound 165 (Kowa Company Ltd., Japan – also known as K-115, originated from D. Western Therapeutics Institute, Japan. Figure 16)249, 254, a close analogue of 28, has been the first ROCK inhibitor approved for the treatment of glaucoma (twice daily, 0.4% eye drop) in Japan249. In a phase 1 trial (randomized, placebo-controlled and double-blinded), the effect of

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165 at doses ranging from 0.05% to 0.8% was assessed in 50 healthy volunteers255. After administration of a single dose, IOP changes from baseline at two hours post-instillation ranged from 2.2 to -4.3 mmHg for 165 vs. -1.6 mmHg for placebo, with maximal and significant changes found for the groups dosed with 0.4% and 0.8% (respectively -4.0 and -4.3 mmHg from baseline / -2.4 and -2.7 mmHg from placebo at two hours). The effect was also significant at the 0.05% dose (-1.8 mmHg vs. placebo) but not at the 0.1% and 0.2% groups. Conjunctival hyperemia was a common adverse event in all groups being observed in 25% of the 0.2% group and in at least 50% of the other groups. Repeated dosing (twice daily for 7 days) confirmed the significant IOP reduction compared to baseline but not placebo. In a phase 2 trial, Tanihara and colleagues have shown that 0.2% and 0.4% 165 dosed twice daily for 8 weeks decreased IOP in the range of 2 mmHg (vs. placebo) 2 hours post-instillation, while incidence of hyperemia was 43%, 57% and 65% respectively for 0.1%, 0.2% and 0.4% 165234. In another phase 2 trial, the same group has monitored the IOP decrease over 24 hours in patients receiving 0.2% and 0.4% 165 (twice daily) and showed that maximal IOP reduction was between 2.9 and 4.4 mmHg (at 2 two hours after first and second instillation). However, IOP decrease was no longer significant at 10 to 12 hours after administration, confirming that twice daily administration is required to achieve IOP reduction over 24 hours256. More recent clinical data from phase 3 trials have been presented at the World Ophthalmology Congress in Tokyo257, 258. Twice daily administration of 0.4% 165 over 8 weeks resulted in a mean IOP lowering of 3.4 mmHg (4 mmHg peak effect) compared to baseline. Transient conjunctival hyperemia was again a very common adverse event, affecting almost 74% of the patients (vs. 1.9% for placebo). The effect of concomitant administration of 0.4% 165 with either

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latanoprost (0.005% once a day, evening dosing) or timolol (0.5%, twice daily administration), was found to induce a greater reduction in IOP than latanoprost or timolol monotherapy (2.7 and 2.6 mmHg additional vs. baseline respectively). Long-term safety and efficacy of 165, either alone or in combination therapy was evaluated in a 52 weeks trial which evaluated monotherapy, concomitant administration with latanoprost or timolol or the combination of both. It is unlikely that a fixed-dose combination of 165 with either latanoprost or timolol represents a viable therapeutic option. Indeed, latanoprost is typically given once daily, while 165 requires twice daily administration in order to maintain 24 h IOP control. ROCK inhibitors have also been shown to reduce the ocular bioavailability of timolol, thereby making this therapeutically inappropriate259. Aerie Pharmaceuticals has also developed ROCK inhibitors for the treatment of glaucoma. AR-12286 (structure not disclosed) was the first ROCK inhibitor from Aerie to enter clinical development back in 2009. This molecule originated from the 6-amido-isoquinoline amide series exemplified by AR-12141 (166)260, 261. Following encouraging IOP lowering effect in animal models, AR-12286 was evaluated in a number of clinical trials (ClinicalTrials.gov identifiers NCT00902200, NCT01330979, and NCT01699464). In normotensive volunteers, two different formulations (both 0.5% AR-12286, once daily) were tested and demonstrated up to 7 mmHg IOP decrease compared with baseline262. In another dose-ranging study in glaucomatous or ocular hypertensive patients, administration of 0.05%, 0.1% and 0.25% AR-12286 once or twice daily administration was evaluated. The once daily administration involved 7 days morning dosing and then 7 days evening dosing. IOP-lowering effects were found in all concentrations, with twice daily administration of 0.25% AR-12286 giving the largest decrease in IOP (up to -6.8 mmHg compared to baseline)263. The only reported adverse effect was mild to moderate hyperemia lasting for maximum 4 hours. In spite of these encouraging results, a subsequent trial indi-

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cated that the effects decreased over time (-6.7 mmHg at day 7 and -5.3 mmHg at day 28), and thus development of AR-12286 for the treatment of glaucoma was discontinued in 2013. In the meantime, Aerie has been developing a fixed-dose combination of AR-12286 and travoprost (a marketed prostaglandin analogue). In a phase 2a study, the combination of 0.5% AR12286/0.004% travoprost was shown to lower IOP in glaucoma patients by approximately 2 mmHg on top of the IOP lowering effect of 0.004% travoprost as monotherapy. Conjuctival hyperemia was again a very common adverse event affecting 59% of patients. This fixed-dose combination has also been discontinued. AR-13324 (structure not disclosed) is another ROCK inhibitor currently in phase 3 clinical trials in the US and Canada. This molecule is also derived from the 6-amido-isoquinoline series originating from AR-12432 (167) but, in contrast to AR-12286, it is rapidly degraded in vivo into a more active metabolite (AR-13503, structure not disclosed)260, 261, 264. In addition to inhibiting ROCK-I and ROCK-II, it was reported that AR-13324 and AR-13503 also inhibited PKCθ and MRCK-α (Table 3) as well as norepinephrine transporter (NET, Ki = 410 nM)261. It is not yet fully understood how all these activities together contribute to the pharmacodynamic effect on IOP. Interestingly, significant differences between the two enantiomers have been described (AR-13325, structure not disclosed, being to co-enantiomer of AR-13324, Table 3). In normotensive monkeys, 0.04% AR-13324 showed significant IOP reduction (25% reduction compared to baseline and 24% compared to the untreated contralateral eye used as control)196. In addition to its direct effect on IOP, AR-13324 also decreased episcleral vein pressure in Dutch Belted rabbits265. In a phase 1 clinical trial involving normotensive volunteers, administration of 0.02% AR-13324 eye drops once daily for 8 days was found to be safe and well tolerated (with only one subject exhibiting hyperemia 8 hours after dosing) and the baseline IOP was significantly re-

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duced (up to 6 mmHg)195. In a phase 2 trial (ClinicalTrials.gov identifier NCT01731002), 0.01% and 0.02% AR-13324 significantly lowered mean diurnal IOP by 5.5 and 5.7 mmHg respectively, whereas 0.005% latanoprost reduced IOP by 6.8 mmHg. A greater incidence of hyperemia was seen in AR-13324-treated arms (52% and 57% respectively for 0.01% and 0.02%, compared to latanoprost, 16%). This higher incidence of hyperemia was still observed on the day following the last administration at the end of the study (18%, 24%, 11% respectively for 0.01% and 0.02% AR-13324 and 0.005% latanoprost respectively)194. Aerie Pharmaceuticals started clinical phase 3 trials in 2014 with both once daily and twice daily 0.02% AR-13324 using timolol as a comparator (ClinicalTrials.gov identifiers NCT02246764, NCT02207621). In this trial, AR-13324 did not meet the primary endpoints but was shown to be non-inferior to timolol, and a further trial is planned in 2015-2016. Moreover, in 2014 Aerie also completed a phase 2b clinical trial with a fixed-dose combination of 0.02% AR-13324 and 0.005% latanoprost (ClinicalTrials.gov identifier NCT02057575). When taking into account baseline differences between different groups, AR-13324 provided an additional 1 mmHg reduction compared to latanoprost monotherapy. A phase 3 trial with AR13324 has reportedly started in 2015. Finally, Aerie recently disclosed data with AR-13533 (http://www.aeriepharma.com/, structure not disclosed), a second generation of dual ROCK/NET inhibitor. This molecule does not require enzymatic conversion to exert IOP lowering effects. INS117548 (168), developed by Inspire Pharmaceuticals Inc. (acquired by Merck in 2011), is an isoquinoline derivative115, 266 and a potent inhibitor of ROCK-I and ROCK-II (Ki = 5 and 14 nM respectively)267. It was shown to be effective at reducing IOP in monkey eyes (20-25% relative to baseline at 1-3 hr following instillation). In 2008, Inspire initiated a phase 1 trial to assess

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the safety and tolerability of 168 (three doses, twice daily administration) in over 80 patients with bilateral ocular hypertension or early POAG (ClinicalTrials.gov identifier NCT00767793). Detailed data from this trial has not been published but it was reported that 168 showed only mild reduction of IOP and was associated with ocular discomfort (ocular burning and stinging)268. No further development has been reported for this compound. Following a license agreement between Asahi Kasei and Altheos venture (from Asahi Kasei website), Altheos Inc. was set up to further develop ATS907 (structure not disclosed) for glaucoma outside Japan and Korea. ATS907 is a small molecule ROCK inhibitor that undergoes rapid metabolic conversion into a more potent metabolite ATS907M1 (Ki = 36 and 37 nM on ROCK-I and ROCK-II respectively for ATS907, and Ki = 7.5 and 7.8 nM on ROCK-I and ROCK-II respectively for ATS907M1). After topical administration in Japanese White Rabbit (JWR) eyes, ATS907 showed no measurable systemic level of either parent and metabolite269. In 2012, Altheos conducted a phase 2 trial (ClinicalTrials.gov identifier NCT01668524) in patients with POAG (single administration of 3 increasing concentrations). Although there is no reported pharmacodynamic data resulting from this trial, it is likely that the effect on IOP decrease was not sufficient to warrant further development. Meanwhile, in vivo evaluation of another candidate, ATS8535 (structure not disclosed) was initiated. At 2 to 4 hours post administration, a maximum IOP reduction from baseline in the range of 4 to 5 mmHg (0.008% and 0.08% in JWR respectively) and 5 mmHg (0.2% in non-human primate – significantly superior to the effect of latanoprost) was observed270. Subsequently, no further data on any Altheos compounds have been disclosed. Santen (Japan) has also conducted several trials (phase 1/2) with ROCK inhibitor DE-104 (structure not disclosed), but as the endpoints were not reached, this project was discontinued268.

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Compound 34 (originally from Mitsubishi Pharma, then Senju and subsequently Novartis), is a more potent analog of 31. It has been shown to significantly lower IOP in a dose-dependent manner on both rabbit and monkey eyes at concentrations varying from 0.01% to 0.1%107. The IOP decrease resulting from administration of 0.05% 34 was shown to be similar to that of 0.005% latanoprost (in the range of 2.5 mmHg) on monkey eyes. Ocular toxicology studies have subsequently been performed in rabbits (4 weeks, 4, 3 or 2-times a day, doses from 0.003% to 0.03%) and in monkeys (26 weeks, 4 weeks, 4, 3 or 2-times a day, doses from 0.003% to 0.05%) and have not revealed any toxicological irreversible findings. The most common adverse effects in these studies were conjunctival hyperemia and sporadic punctate subconjunctival hemorrhage (in the 4-times daily arm). Subsequently, Senju conducted phase 1/2 clinical trials with 34 eye drops administered twice-daily for 7 days at 0.01% and 0.05% in healthy volunteers. The most common adverse event again was bulbar conjunctival hyperemia (in 5 patients out of 6 for both concentrations)271. Another trial was conducted to compare the effect of 34 0.05% and 0.1% for 29 days vs. vehicle and 0.005% latanoprost in glaucoma patients, but it is our understanding that further development has been discontinued due to tolerability issues. AMA0076 (structure not disclosed) is a potent, selective and soft ROCK inhibitor from a recently reported series of 3-[2-(aminomethyl)-5-[(pyridin-4-yl)carbamoyl]phenyl] benzoates217, 233. In vitro, AMA0076 significantly inhibited actin stress fibers and focal adhesions in human trabecular meshwork cells. In normotensive New Zealand White (NZW) rabbits, both 0.1% and 0.3% AMA0076 induced a significant lowering of IOP while causing only very mild hyperemia. In an acute hypertensive model in NZW rabbits, AMA0076 was shown to significantly prevent the rise in IOP induced by viscoat® injection. In both cases, AMA0076 was found to be more effective than latanoprost (and superior to bimatoprost in the hypertensive model). AMA0076 was subse-

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quently evaluated in two clinical trials272. In the first in-human trial (multicenter, double blinded, placebo-controlled), a first formulation of AMA0076 was administered twice daily in 82 POAG/OHT patients in a dose-escalation manner. The most effective dose achieved a decrease in mean diurnal IOP from baseline of 3.7 mmHg (compared to placebo), while no significant hyperemia was reported. Alternative formulations of AMA0076 have been evaluated in a phase 1b study (single center, randomized, double-masked, placebo-controlled, repeat-dose, 3 period cross over study) in 21 ocular normotensive volunteers (14 active and 7 placebo). The tested formulations were administered twice daily for 1 week, with a 1-week washout period between treatments. In this study, AMA0076 was well tolerated both systemically and locally (only mild and transient hyperemia was reported in a minority of the subjects) and was shown to significantly lower the mean IOP. Amakem subsequently commenced a phase 2a trial, but data have not yet been disclosed. 4.2. ROCK inhibitors for the treatment of other ocular diseases. ROCK inhibitors have recently been investigated for their applicability in the treatment of corneal endothelial disorders, a group of disabling ocular disorders with a high unmet medical need. Some initial and limited clinical trials have been conducted with 31 by Kinoshita et al. based on results obtained in both in vitro and in vivo studies. The in vitro studies showed that 31 promoted cell adhesion and proliferation, and inhibited apoptosis of primate corneal endothelial cells in culture. These positive results were confirmed in both rabbit273 and monkey models274 of partial endothelial dysfunction, where corneal endothelial wound healing was accelerated by topical application of 31 to the ocular surface, resulting in regeneration of a corneal endothelial monolayer with a high endothelial cell density. A phase 1 trial with 10 healthy volunteers showed that 31 could be administered as 10 mM eye drops 6 times daily for 7 days without safety concerns. Furthermore, in the pilot clinical

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study, performed at the Kyoto Prefectural University of Medicine, the effects of 31 eye drops after trans-corneal freezing were evaluated in 8 patients with corneal endothelial dysfunction. 31 eye drops proved effective for the recovery of corneal transparency and for gradual reduction of corneal thickness for up to 6 months in human patients with central edema due to endothelial dysfunction274. In one case of a late-onset Fuchs corneal dystrophy, treatment with 31 led to corneal clearing and improved vision 2 weeks after treatment. At 6 months, vision was further improved and central corneal thickness was significantly lower than observed before commencement of treatment275. Further clinical studies are warranted to confirm these results. Although ROCK inhibition may be beneficial for the treatment of multiple other eye diseases, their development in these indications has, to date, been limited by issues with formulations and acceptable frequency of intravitreal injections. Nevertheless, currently available treatments for wet Age-related Macular Degeneration (wet AMD) or diabetic retinopathy (DR) are dominated by anti-VEGF antibodies or steroids with well-known side-effects. In 2014, Kowa disclosed preclinical data showing beneficial effects of 165 in both in vitro and in vivo studies related to diabetic retinopathy276. In vitro, 165 was shown to significantly inhibit the migration and the proliferation of human retinal microvascular endothelial cells (HRVECs) induced by VEGF. In a mouse model of oxygen-induced retinopathy (OIR), 165 (0.4% and 0.8%) was administered topically three times daily for 6 days (postnatal day 12 to 17). Analysis of retinal flatmounts demonstrated a significant decrease in pathological neovascularization, avascularity (23% and 41.9% reduction respectively for 0.4% and 0.8% 165), and fluorescein leakage (58.4% for 0.8% 165). Based on these data and on the clinical safety data generated with this molecule in glaucoma, Kowa has recently initiated a phase 2 clinical trial to evaluate the efficacy of 165 for the treatment of diabetic retinopathy and diabetic macular edema.

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There are at least two other ROCK inhibitors that have very recently shown encouraging results for the treatment of wet AMD. The first molecule, AMA0428 (structure not disclosed) from Amakem is a very potent and selective soft ROCK inhibitor (IC50 < 1 nM against ROCK-II and EC50 < 10 nM in a MLC-phosphorylation assay). In in vitro studies235, AMA0428 was shown to significantly reduce proliferation of human umbilical vein endothelial cells (HUVECs) and human brain microvascular endothelial cells (HBMECs), to inhibit VEGF-induced migration of HBMECs and to stimulate HBVPs migration. In a laser-induced mouse model of CNV, a single intravitreal (IVT) injection with AMA0428 at either day 0 or day 3 resulted in a significant decrease of inflammation in contrast to DC-101 (mouse anti-VEGF-R2). Multiple IVT injections significantly reduced neovascularization and vessel leakage with a magnitude comparable to DC101. In contrast to DC-101, repeated IVT injections decreased inflammation (-31%) and collagen deposition (-43%). More recently, the same molecule was tested in two additional mouse models of diabetic retinopathy: the oxygen-induced retinopathy (OIR) model and the streptozotocin (STZ)-induced diabetic model and the data will be published in the near future. Aerie Pharmaceuticals presented in vivo data of AR-13154 (structure not disclosed), a mixed ROCK-II, JAK2, JAK3 and PDGFR-β inhibitor (inhibition of >99%, 72%, 97%, and 89%, respectively at 500 nM) in a model of laser-induced CNV in rats277. CNV was induced on day 0 and AR-13154 was administered intravitreally on days 1, 4 and 10 (n = 5 per group, at concentration of 0.06, 0.6 and 6 µg/ml), with aflibercept (800µg/ml) on day 1 as positive control. At the end of the study (day 21), lesion size was reduced by 35% for AR-13154 at 6 µg/ml (no effects at other doses) compared with 23% for aflibercept. Aerie Pharmaceuticals has yet to announce any further developments for this molecule.

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A study to evaluate the effect of 28 combined with bevacizumab for the treatment of diabetic macular edema (DME) is also listed on clinicaltrials.org (ClinicalTrials.gov identifier NCT01823081). In this study, 15 patients with severe DME who received concomitant injections of bevacizumab and 28 (0.025 mg/0.05 ml), significantly improved visual acuity and decreased central macular thickness, confirming results seen in a previous small trial278, 279. 4.3. ROCK inhibitors in cancer. Although the use of ROCK inhibitors in oncology has been well described in literature, the potential systemic side-effects such as decrease of blood pressure has, to date, limited their study as systemic therapies in cancer. According to the available literature, there is currently only one ROCK inhibitor, AT13148 (169)172, in clinical development for oncology indications. Using a fragment-based approach, Astex (UK) has optimized a series of pyrazole derivatives172 and eventually selected 2-amino-1-(4-chloro-phenyl)-1-[4-(1H-pyrazol-4yl)-phenyl]-ethanol as a lead compound. Subsequently, it was found that the S-enantiomer of this compound was significantly more potent on some AGC kinases including ROCK and AKT280. Indeed, rather than a selective ROCK inhibitor, 169 is actually an inhibitor for multi-AGC kinases affecting ROCK-I, ROCK-II, p70S6K, PKA, AKT1, 2 and 3, RSK1, and SGK3 (Table 4)281. In vitro, 169 was shown to potently inhibit the phosphorylation of the respective substrates of these kinases and to induce apoptosis in relevant cancer cells. The oral bioavailability of 169 is high, and exposure has been found to be proportional with the administered dose. In different animal xenografts models, 169 was found to have a significant effect in reducing the tumor volume at doses of 40 or 50 mg/kg (oral administration). Interestingly, it is noted that 169 showed cardiovascular side effects including vascular smooth muscle contraction, reduction of blood pressure and tachycardia, but that these effects resolved after repeated dosing. Detailed data were not provided. Following these results, Astex and the Institute of Cancer Research (UK) initiated

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a phase 1 clinical trial in 40 patients with solid tumor (ClinicalTrials.gov identifier NCT01585701). Data are expected in mid-2015. Scientists from Moffitt Cancer Center are currently evaluating the potential of 170, a pyridine-thiazole urea based ROCK inhibitor derived from a series of pyridine-thiazole amide282. 170 showed an IC50 of 14.5 and 6.2 nM on ROCK-I and ROCK-II respectively, potently inhibited phosphorylation of the ROCK substrate MYPT1 and showed excellent in vivo efficacy in an animal model of breast cancer53,

117

. Further development of 170 has however not been an-

nounced. Despite numerous studies to further understand the role of ROCK in oncology, according to the literature, no other ROCK inhibitors are currently in clinical development for the treatment of cancer. 4.4. ROCK inhibitors for the treatment of erectile dysfunction (ED). In the past few years, Sanofi filed more than 12 patent applications on ROCK inhibitors based on the isoquinoline / isoquinolinone scaffold and eventually selected 171 (Figure 16) for further profiling and clinical evaluation283, 284. 171 is a potent, selective and ATP-competitive ROCK inhibitor (Ki = 36 nM in human recombinant ROCK)202. In vitro, this compound was shown to block thrombin-induced stress fibers formation, to inhibit significantly the proliferation of human aortic smooth muscle cell and the migration of inflammatory cells. Interestingly, in hypertensive rats, oral administration of 171 (1, 3, 10 and 30 mg/kg) significantly decreased blood pressure at all doses (with associated tachycardia only for the highest dose) whereas the blood pressure was only decreased significantly at the highest dose in normotensive rats (associated with tachycardia as well). No difference in plasma levels between strains was found, suggesting a potential differentiation between a normal vs. diseased state. The ability of 171 to lower blood pressure in animal models of

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hypertension was subsequently confirmed. More recently, 171 was shown to reduce renal vascular resistance in animal models as well as in human tissues203. More intriguingly, it was superior to sildenafil in the induction of penile erection in normal rabbits (after iv or po administration) and in diabetic rabbits (po administration) both in intensity and in duration of action, as well as in preparations of phenylephrine pre-contracted rat and human corpus cavernosum205. In 2009 and 2011, Sanofi conducted clinical trials with a single dose trial in patients with mild to moderate erectile dysfunction (ClinicalTrials.gov identifier NCT00914277) and in patients with moderate chronic kidney disease (ClinicalTrials.gov identifier NCT01485900). According to the available literature, further development has been discontinued in these fields. In 2013, Sanofi announced that 171 would be evaluated in pulmonary hypertension and additional data on hypertension were reported recently204. However, this compound is no longer listed on Sanofi’s R&D portfolio (http://en.sanofi.com/rd/rd_portfolio/rd_portfolio.aspx, accessed on June 15, 2015). Similarly to the study by Guagnini et al., 28 was also shown to restore erectile function in diabetic rats, but no clinical trial has been reported to date285. 4.5. ROCK inhibitors for the treatment of other diseases. After its acquisition of Surface-Logix Inc. in 2011, Nano Terra Inc. entered into a license agreement with Kadmon the same year under which Kadmon received worldwide rights to various assets including 45. As described above, 45 is currently the only ROCK-II specific inhibitor that has achieved significant selectivity for ROCK-II versus ROCK-I which, in itself, is believed to improve the safety window compared to pan-ROCK inhibitors. In January 2015, Kadmon announced the initiation of a 3 month phase 2 trial using 45 (200 mg twice daily and 400 mg once daily) in patients with moderate to severe psoriasis (ClinicalTrials.gov identifier NCT02317627). In a phase 2a trial, administration of 200 mg 45 showed promising efficacy with reduction by up to 66% of the Psoriasis Area and Severi-

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ty Index (PASI) scores in 3 of 8 patients. Kadmon also plans to evaluate 45 in systemic lupus erythematosus, iodiopathic pulmonary fibrosis (IPF) as well as in chronic graft-versus-host disease. Further disclosure of the clinical data will support a better understanding of the safety issues with ROCK inhibitors and help elucidate the role of the ROCK-II specific isoform. Compound 28 has been successfully used in small trials in both CNS diseases286 and cardiovascular conditions287. Over the past few years, many ROCK inhibitors with greater potency and selectivity than 28 have been developed. These molecules have showed promising efficacy in a variety of animal models in the fields including pain, respiratory (such as IPF288) and cardiovascular diseases. Although data need to be confirmed in a clinical setting, these encouraging findings pave the way for further investigations. In conclusion, over recent years the clinical evaluation of ROCK inhibitors has been the most focused in the reduction of IOP in glaucoma. Despite hyperemia and sometimes limited IOP control, one compound (165) has now reached the market in Japan and another compound is in Phase 3 clinical development (RhopressaTM, Aerie Pharmaceuticals). In the context of glaucoma, the benefits of inhibiting ROCK might go beyond the recognized IOP-lowering effects, as for example shown by Tokushige et al. and Yamamoto et al., who reported that 34 and 165 may both have neuroprotective effects108, 289. However, as hyperemia has so far severely limited the long-term use of ROCK inhibitors, this aspect has yet to be confirmed clinically. For applications outside the field of ophthalmology, only a limited number of ROCK inhibitors have so far entered clinical development despite numerous reports of their beneficial effects in various animal models, most probably as a result of their narrow therapeutic window. 5. Conclusions and Future Considerations

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In this Perspective, over 170 ROCK inhibitors are reviewed. Their chemical series include isoquinoline/isoquinolinone, indazole, pyridine, pyrimidine, pyrrolopyridine, pyrazole, benzimidazole, benzothiazole, benzathiophene, benzamide, phthalazinone, aminofurazan, quinazoline, and boron derivatives. Many of these ROCK inhibitors are highly potent with IC50 values in the nanomolar range and with good physicochemical and pharmacokinetic properties. Some of these molecules demonstrated high kinase selectivity with a hit ratio of ~ 1% against the Ambit kinase panel (74, 78), and a few showed good isoform selectivity (> 50-fold) of ROCK-II vs. ROCK-I. Compounds with ROCK-II isoform selectivity, such as 45, 154, and 155, might have the advantage of fewer side effects. Optimization of pharmacokinetic properties by reducing systemic exposure is another recent and interesting development in the field of ROCK inhibitors. Developing ROCK inhibitor based on the soft drug approach represents an attractive alternative. Compound 28 is used in the treatment of cerebral vasospasm in Japan and China. However, 28 has a moderate potency with a Ki of 330 nM against ROCK-II and, more importantly, has inhibitory activity against a number of other kinases in the AGC family. Isoform selectivity might be crucial to minimize unwanted ROCK-I inhibition. Once desired selectivity has been accomplished, ROCK-II specific inhibitors may potentially be used in CNS disorders such as Alzheimer’s disease. All 13 X-ray co-complex structures reported are type-I ROCK inhibitors. For future ROCK inhibitor development, discovery of type-II (DFG-out), type-III (allosteric) and type-IV (covalent) ROCK inhibitors would be interesting but have yet to be described. Both type-II and type-III are more selective than type-I, and therefore may have greater potential to improve selectivity and thus improve the therapeutic index and drug safety.

Author Information

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Corresponding Authors For Y.F.: phone, 561-228-2201; E-mail, [email protected]. For R.L.: phone, 402-559-6609; fax, 402-559-5418; E-mail, [email protected]. Notes The authors declare no competing financial interest. Biographies Yangbo Feng is Associate Scientific Director of Medicinal Chemistry / Drug Discovery at the Translational Research Institute of the Scripps Research Institute. Dr. Feng has been in charge of the medicinal chemistry efforts for several kinase and non-kinase inhibitor development projects including ROCK. Before joining Scripps in 2004, he was a Senior Scientist and group leader at Discovery Partners Internationals (now part of BioFocus) working on a variety of projects targeting proteases, GPCRs, ion-channels, and kinases. Dr. Feng received his PhD in bioorganic chemistry from the University of California San Diego and obtained his postdoctoral training under the guidance of Dr. Kevin Burgess at Texas A&M University. Yangbo is co-inventor of more than 20 patents / patent applications, and co-author of more than 50 peer reviewed manuscripts. Philip LoGrasso is a Professor and Senior Director of Drug Discovery in The Scripps Research Institute. Dr. LoGrasso has been in charge of the discovery biology efforts for several drug discovery projects including ROCK. Before joining Scripps in 2005, he was the Director of preclinical research and development at Avera Pharmaceuticals, a CNS drug development company focused on neurology and psychiatry. Prior to joining Avera, Dr. LoGrasso was a Research Fellow at Merck for nine years working in the areas of inflammation and molecular neuroscience. Dr. LoGrasso received his Ph.D in Pharmacology in 1992 at the University of Florida and

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did postdoctoral training at the Sandoz Research Institute. Phil is co-inventor of 15 patents / patent applications, and co-author of more than 80 peer reviewed manuscripts. Olivier Defert is co-founder of Amakem and has managed R&D chemistry and regulatory preclinical development since 2010. Olivier led the preclinical development of AMA0076, a ROCK inhibitor in phase 2 for the treatment of glaucoma and ocular hypertension. Prior to Amakem, Olivier was section leader in Medicinal Chemistry at Devgen NV in Belgium for 8 years, with responsibilities in combinatorial chemistry, medicinal chemistry and preclinical development of the Devgen’s lead ROCK inhibitor for the treatment of Crohn’s disease and PKC inhibitor for the local treatment of psoriasis. He received his PhD in medicinal chemistry from the Faculty of Pharmacy in Lille (France) in 2005. He is co-inventor of more than 20 patents/patent applications and co-author of 4 peer reviewed publications. Rongshi Li is a Professor of Chemistry and Medicinal Chemistry, Department of Pharmaceutical Sciences, University of Nebraska Medical Center (UNMC). He received his Ph.D. from Dalhousie University in Canada. After postdoctoral studies with George L. Kenyon at University of California San Francisco and K.C. Nicolaou at IRORI in San Diego, he spent 14 years in industry advanced from Scientist to Senior Vice President. Dr. Li began his academic career in 2008 at Moffitt Cancer Center. In 2013, he was recruited to the Center for Drug Discovery, Department of Pharmaceutical Sciences, and Fred and Pamela Buffett Cancer Center, UNMC. Since 2008, Dr. Li has published over 20 peer-reviewed articles, filed and published 28 US and PCT patents, edited two books, and delivered over 30 invited talks. Acknowledgements We thank Drs. Sandro Boland and Kieran Rooney for reviewing the manuscript.

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Abbreviations Used AD, Alzheimer’s disease; AGC, cAMP-dependent protein kinase/protein kinase G/protein kinase C; AIA, adjuvant induced arthritis; AMD, age-related macular degeneration; ATP, adenosine triphosphase; BP, blood pressure; CNS, central nervous system; COPD, chronic obstructive pulmonary disease; CRMP2, collapsing response mediator protein-2; CYP450, cytochrome P-450; DME, diabetic macular edema; DMPK, drug metabolism and pharmacokinetics; ED, erectile dysfunction; EOB, eye open at birth; ERM, ezrin-radixin-moesin; GAP, GTPase activating protein; GDP, guanosine diphosphate; GEF, guanine nucleotide exchange factors; GTP, guanosine triphosphate; HBMECs, human brain microvascular endothelial cells; hERG, human-either-a-gogo; HPV, hypoxic pulmonary vasoconstriction; HTS, high throughput screening; HUVECs, human umbilical vein endothelial cells; IOP, intraocular pressure; OIR, oxygen-induced retinopathy; IVT, intravitreal; MARCKS, myristylated alanine-rich C-kinase; MEFs, mouse embryonic fibroblasts; MIA, monoiodoacetate induced arthritis; MLC, myosin light chain; MLCP, myosin light chain phosphate; MS, multiple sclerosis; NADPH, the reduced form of nicotinamide adenine dinucleotide phosphate; NAMPT, nicotinamide phosphoribosyltransferase; NF-L, neurofilament protein; NHE1, adducing, sodium/hydrogen exchanger 1; NOX, NADPH oxidase; OA, osteoarthritis; OIR, oxygen-induced retinopathy; PCT, Patent Coorperation Treaty; PK, pharmacokinetics; PKA, protein kinase A; PKB, protein kinase B; POAG, primary open-angle glaucoma; ROCK, Rho kinase; ROS, reactive oxygen species; SAR, structure activity relationship; SCI, spinal cord injuries.

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R

Ra O

H2N

OH B O

Rb

F O H2N 131, IC50: ROCK-II: 540 nM ROCK-I: 810 nM

130, Boron based ROCK inhibitors F O H2N

OH B O

Cl

OH B O

OH B O

O H2N

132, IC50: ROCK-II: 220 nM ROCK-I: 250 nM Cl O H2N 134, IC50: ROCK-II: 47 nM ROCK-I: 59 nM

133, IC50: ROCK-II: 340 nM ROCK-I: 730 nM OH B O

O

OH B O

135 -lactamase inhibitor

Figure 12. Boron derivatives as ROCK inhibitors

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Table 1. DMPK properties for ROCK inhibitors derived from the chroman amide, urea, and quinazolinone scaffolds. cmpd Microsomal stability (T1/2, min) CYP450 % inh. at 10 µM Cmaxa Cla AUCa t1/2a V da Human Rat 1A2/2C9/2D6/3A4 µM mL/min/kg µM.h h L 47.5 6.5 35/96/88/6 3.7 8.2 5.9 3.0 1.7 107 62.5 16.5 -3/8/3/9 27.9 0.7 68 2.7 1.4 108 77.2 7.8 -10/2/-12/8 43.3 0.25 167 5.0 0.1 109 64.3 3.4 10/61/3/8 2.6 16.3 2.2 1.4 1.3 110 69.0 3.5 -8/77/63/3 1.0 19.2 1.9 2.3 3.6 111 46.1 7.3 -2/46/77/8 1.5 23.0 1.2 1.0 1.7 75 128.9 24.4 9/92/29/15 1.1 30.9 3.6 1.9 1.9 82 23.7 15.1 -23/95/38/37 3.8 11.2 4.2 1.6 1.0 83 a i.v. dosing on rats at 1 mg/kg; b p.o. dosing at 2 mg/kg on rats.

Table 2. Clinical development status of ROCK inhibitors Cmpd

Organization

Therapeutic application

Development Status

28

Asahi Kasei

Cerebral vasospam

Approved (Japan & China)

165

Kowa

Glaucoma Diabetic retinopathy

Approved (Japan) Phase 2

AR-13324

Aerie

Glaucoma

Phase 3

AR-12286

Aerie

Glaucoma

Phase 2 - Discontinued

PG286*

Aerie

Glaucoma


PG324**

Aerie

Glaucoma

Phase 2

34

Senju/Novartis

Glaucoma


ATS907

Altheos

Glaucoma


AMA0076

Amakem

Glaucoma

Phase 2

DE-104

Santen

Glaucoma


168

Inspire (Merck)

Glaucoma


31

University of Kyoto

Corneal endothelial disorders

Phase 1/2 Phase 2 Phase 2 Phase 2 Phase 2 Phase 2

45

Kadmon

Psoriasis Idiopathic Pulmonary Fibrosis Nonalcoholic Steatohepatitis Lupus Nephritis Graft versus Host Disease

169

Astex

Solid tumors

Phase 1

171

Sanofi-Aventis

Erectile dysfunction Chronic kidney disease

Phase 2 – Discontinued Phase 1 - Discontinued

*: AR-12286 in combination with travoprost; **: AR-13324 in combination with latanoprost.

109


Fb % 79 46 26 3 0 35 46 51


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Table 3. Inhibitory activities of AR-13324, AR-13325, and AR-13503 (IC50 in nM) Kinase

AR13324

AR13325

AR13503

ROCK-I

1.2

68

0.2

ROCK-II

1.1

91

0.2

MRCKα

129

1843

7

PKCθ

92

5383

27

Table 4. Inhibitory activities of 169 Kinase

IC50 (nM)

ROCK-I

< 10

ROCK-II

< 10

P70S6K

< 10

PKA

< 10

AKT1

38

AKT2

402

AKT3

50

RSK1

85

SGK3

50

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TOC Graphic

GEF GDP

Rho

NH

NH

GTP

O N FO S

O N O S

Rho ROCK

GAP

N

N

MLCP MLC

LIMK

MARCKS

NF-L

111

CRMP2


Adducin

NHE1

ERM

Rho Kinase (ROCK) Inhibitors and Their ... - ACS Publications

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